Vaccination with mica/b alpha 3 domain for the treatment of cancer

ABSTRACT

The present invention provides compositions and methods for treating cancer in a subject by eliciting an immune response against a MIC alpha 3-domain polypeptide.

RELATED APPLICATIONS

This application is a divisional application of Ser. No. 15/781,448,filed on Jun. 4, 2018, which is a U.S. National Phase application, filedunder 35 U.S.C § 371, of International Application No.PCT/US2016/064969, filed on Dec. 5, 2016, which claims priority to andbenefit of U.S. Provisional Application No. 62/263,377, filed on Dec. 4,2015, and 62/422,454, filed on Nov. 15, 2016, the contents of each ofwhich are hereby incorporated by reference in their entireties.

GOVERNMENT INTEREST

This invention was made with government support under R01CA173750awarded by the National Institutes of Health. The government has certainrights in the invention.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named 5031461-080US4_SL.txt, which wascreated on May 25, 2021 and is 32,794 bytes, are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to composition and methods forinducing an anti-tumor immune response in a subject.

BACKGROUND OF THE INVENTION

Recent advances in the field of cancer immunotherapy have demonstratedthe ability of our immune system to eradicate even advanced cancers.These therapies are rapidly changing the face of cancer treatment.Unlike monoclonal antibody therapies which require repeatedadministration of antibodies to prevent tumor relapse, vaccines caninduce endogenous immunological memory and thus have the potential toprovide long-term protection.

The selection of antigens for vaccine therapy requires a comprehensiveunderstanding of the biological role of the candidate antigens in tumorgrowth and their expression levels by tumor cells compared to normaltissues. MICA and the closely related MICB protein (abbreviated as MIC)are antigens that are absent or expressed at very low levels by normalcells, but are broadly upregulated by a variety of different cancerssecondary to genomic damage. MIC is an important ligand for the NKG2Dreceptor on cytotoxic lymphocytes, specifically NK cells, CD8 T cellsand gamma-delta T cells. Expression of MIC targets such cells forelimination by the immune system. However, many tumors are found toescape this important immune surveillance pathway by shedding MIC fromthe cell surface, a process in which the MIC alpha3 domain is unfoldedby the disulfide isomerase ERp5 rendering it sensitive to cleavage bymatrix metalloproteases such as ADAM 10 and ADAM 17. Shed MIC causesdownregulation of the NKG2D receptor on NK cells and CD8 T cells.Proteolytic cleavage thus turns an immune-stimulatory protein into animmunosuppressive sub stance.

Thus a need exists for compounds to inhibit MICA shedding.

SUMMARY OF THE INVENTION

In various aspects, the invention provides vaccine compositionsincluding as an immunogenic component, an effective amount of a peptideincluding the MIC alpha 3-domain. By effective amount means an amounteffective to elicit an immune response against the MIC alpha 3-domain.

The MIC alpha 3-domain is a MICA or MICB alpha 3-domain. Optionally, theMIC alpha 3-domain is non glycosylated. Preferably, the peptide includesamino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In various aspects,the vaccine composition comprises a plurality of peptides. In someaspects, the peptide is conjugated to a carrier protein.

In another aspect, the invention provides a fusion protein having amonomeric ferritin subunit protein joined to a MIC alpha 3-domainprotein. The monomeric ferritin subunit protein has a domain that allowsthe fusion protein to self-assemble into nanoparticles. In a preferredembodiment, the monomeric subunit is a Helicobacter pylori ferritinprotein. Optionally, the fusion protein is further conjugated to a CpGoligonucleotide.

In yet another aspect, the invention provides a nanoparticle includingthe fusion protein according to the invention. The nanoparticle includesa plurality of MIC alpha 3-domain peptides.

In yet another aspect, the invention provides a vaccine compositioncomprising the nanoparticle according to the invention. The vaccinecomposition can further comprise GM-CSF.

In a further aspect the invention provides method of treating cancer ina subject by administering to a subject a vaccine composition accordingto the invention. Optionally, the vaccine composition contains GM-CSF.The subject has tested positive for shed MIC in their serum. The vaccinecomposition is administered as part of a therapeutic regimen. Atherapeutic regimen includes for example, radiation therapy, targetedtherapy, immunotherapy, or chemotherapy. Optionally, the subject isfurther administered one or more vaccines specific for an antigen otherthan a MIC alpha 3-domain antigen.

In another aspect the invention provides a method for treating cancer byadministering to the subject a vaccine comprising cells that express MICalpha-3 domain. In a further aspect the invention provides a method fortreating cancer where an immune response against MIC is induced by useof a replicating or non-replicating virus.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

Other features and advantages of the invention will be apparent from andencompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic that demonstrates the interaction between NKG2Dhomodimer and MICA. The MICA-alpha3 domain is identified for reference.From: Nat Immunol. 2001 May; 2(5):443-51. Complex structure of theactivating immunoreceptor NKG2D and its MHC class I-like ligand MICA.

FIG. 1B is a schematic that demonstrates one mechanism through whichtumors escape immune surveillance through the shedding of MIC from thetumor cells' surface.

FIG. 1C is a schematic that depicts a ferritin particle.

FIG. 1D is a schematic that depicts a map demonstrating cellular andhumoral immune responses against human and mouse ferritin.

FIG. 2A is a schematic of the MICA alpha3 ferritin fusion geneconstruct.

FIG. 1A discloses “6 His tag” as SEQ ID NO: 31.

FIG. 2B is (Left) a size exclusion chromatogram of MICA alpha3-ferritinusing XK16/60 Superdex200 column (Flowrate: 2 ml/min; Running buffer: 50mM Tris, 150 mM NaCl pH 7.5). FIG. 2B further depicts (Right) an SDS gelunder reducing conditions containing samples that were collected between22 to 27 minutes at the protein peak.

FIG. 2C is a schematic of the deglycosylated MICA alpha3 construct.

FIG. 2D is (Left) a size exclusion chromatogram of MICA alpha3 usingSuperdex200 column (Flowrate: 1 ml/min; Running buffer: 50 mM Tris, 150mM NaCl pH 7.5). FIG. 2D further depicts (Right) an SDS gel underreducing conditions containing samples that were collected between 16 to20 minutes at the peak protein.

FIG. 3 is a schematic that depicts the use of Mesoporous silica rods(MSR) vaccine for subcutaneous injection, and the resulting induction ofpotent immune responses. See Kim, J & Aileen, W. L. et al. NatureBiotech. 2015.

FIGS. 4A and 4B are a series of graphs that depict the efficacy of MICα3 domain vaccine in a lung metastasis model. The data presented in FIG.4A was obtained as follows. B6 mice were immunized with 200 μg of MIC α3protein, 1 μg of GM-CSF and 100 μg of CpG-ODN, either as a bolus withoutscaffold (bolus) or within the mesoporous silica rods (MSR) scaffold(MSR vaccine). Mice received one booster injection on day 28. Threeweeks later, mice were challenged by i.v. injection of 5×10⁵ B16-MICtumor cells. The number of lung metastases was quantified on day 14following tumor cell injection. The data obtained in FIG. 4B wasobtained as follows. Shed MIC was quantified by ELISA on days 0, 5, and13 following tumor cell injection. Prior experiments had shown that MICα3 domain specific antibodies do not interfere with ELISA used to detectshed MIC.

FIGS. 5A and 5B are a series of plots and graphs that demonstratevaccination with MICA-ferritin fusion protein induces high-titers ofMICA specific antibodies. FIG. 5A depicts a FACS plot of MICAα3 specificantibodies in the sera of immunized mice to full length MICA expressedon the surface of B16F10 mouse melanoma cells. B16F10 melanoma cellswere transfected with human MICA cDNA (allele 009) and then labeled withisotype control antibody (negative control) or a saturatingconcentration of a murine mAb specific for MICA (6D4, positive control).This system was then used to test sera from mice vaccinated with MICAα3—ferritin or a control antigen (OVA). A PE-labeled secondaryanti-mouse IgG antibody was used to detected antibodies bound to thecell surface. Fluorescence was quantified by FACS. Strong staining wasdetected even with 1 μl of serum from mice on days 14-42 followingvaccination. FIG. 5B depicts a bar graph of the mean fluorescenceintensity (MFI) representing +/−SD of 3 replicates of binding of MICAα3specific antibodies in the sera of immunized mice to full length MICAexpressed on the surface of B16F10 mouse melanoma cells.

FIG. 6 is a series of graphs that depict tested sera from MICA-ferritinimmunized mice that were assayed by ELISA to determine the differentsubclasses of IgGs induced upon vaccination.

FIG. 7 is a graph demonstrating that serum antibodies in theMICA-ferritin immunized group prevent MICA shedding from the tumor cellsurface.

FIGS. 8A and 8B are a series of graphs that depict the therapeuticactivity of MICA-ferritin vaccine. C57BL/6 mice were immunized with MICAα3—ferritin or ovalbumin and received a booster injection on day 28.Mice were challenged by intravenous injection of 5×10⁵B16-MICA tumorcells which form lung metastases. The number of lung metastases werecounted on day 14 (FIG. 8A) and shed MICA was quantified in the serum(FIG. 8B). The MICA α3 domain vaccine substantially reduced the numberof lung metastases while the control vaccine had no effect. Also, shedMICA became undetectable in the serum of mice that had received the MICAα3—ferritin vaccine while shed MICA levels were very high in bothcontrol groups. FIG. 8A demonstrates that immunization with MICAalpha3-ferritin prevents metastasis in comparison to naïve controls, oranimals that received OVA-protein injection. FIG. 8B is a graph thatdemonstrates results obtained by ELISA which indicate that vaccinatedmice had undetectable levels of sMICA (shed MICA) in the sera.

FIGS. 9A-9C are a series of graphs that depict the titer of antibodiesinduced by the MICA-ferritin vaccine (FIG. 9A) as well as the effect ofthe vaccine dosage on the number of pulmonary nodules (FIG. 9B) and inthe amount of sMICA (FIG. 9C).

FIGS. 10A and 10B are a series of graphs that depict the effect of thedeglycosylated version of the MICAα3 vaccine (not linked to ferritinnanoparticle) on binding of MICAα3 specific antibodies in the sera ofimmunized mice to full length MICA expressed on the surface of B16F10mouse melanoma cells tested by Flow Cytometry (FIG. 10A), as well asgraphs that depict results from ELISA assays that were used to determinethe different subclasses of IgGs induced upon vaccination (FIG. 10B).

FIGS. 11A and 11B are a series of graphs that depict MICAα3 vaccinealone (without ferritin fusion) has significant therapeutic benefit invivo. FIG. 11A is a graph that depicts the number of pulmonarymetastases following vaccination with MICAα vaccine alone. FIG. 11B is agraph that depicts the amount of sMICA in the serum at day 0, day 5 andday 13 post vaccination with MICAα vaccine alone.

FIGS. 12A and 12B show that MICA-ferritin vaccine delays tumor growth inB16F10 subcutaneous melanoma model. In FIG. 12A, 7 week old C57BL/6female mice (n=8) were immunized with MICA-ferritin vaccine and boostedon day 12. The mice were challenged with subcutaneous injection of0.5×10⁶B16F10 cells expressing MICA on day 25 after initial vaccinationand the tumor volume was measured every other day. Tumor growth in theMICA-ferritin immunized group was found to be significantly slower(empty square) compared to the naïve, untreated age matched controlgroup (filled circle). In FIG. 12B, sMICA levels were undetectable insera of mice immunized with MICA-ferritin vaccine (empty triangle) whilehigh levels of sMICA were detected within two weeks after tumorchallenge in the sera of the non-immunized control group (filledtriangle).

FIGS. 13A and 13B show that depletion of CD8 T cells accelerates tumorgrowth in MICA-ferritin vaccinated B16F10 subcutaneous melanoma model.In FIG. 13A, 7 week old C57BL/6 female mice were immunized withMICA-ferritin vaccine (n=16) or with OVA control vaccine (n=8) andboosted on day 14. The mice were challenged with subcutaneous injectionof 0.5×10⁶B16F10 cells expressing MICA on day 21 after initialvaccination. Mice received intravenous injection of 200 μg of anti-CD8antibody (n=8) or isotype control antibody (n=8) 2 days prior to tumorchallenge and twice a week thereafter at a dose of 100 μg per mouseuntil the study endpoint. Tumor volume was measured every other day. Themice were euthanized when the tumors reached ≥250 mm². Tumors reachedtheir maximum volume by day 12 in OVA protein vaccinated control micetreated with CD8 antibody (empty triangle) and by day 14 in naïve,untreated, non-depleted control group (filled circle). CD8 depletionaccelerated tumor growth in MICA-vaccinated group (filled triangle)compared to MICA-vaccinated group that received isotype antibody (emptysquare). In FIG. 13B, survival analysis of CD8 depletion experimentshowing age matched naïve, untreated, non-depleted control group inthick solid line, OVA protein vaccinated group in thin dashed line,MICA-ferritin vaccinated, CD8 depleted in thick dashed line andMICA-ferritin vaccinated, isotype antibody injected mice in thin solidline.

FIGS. 14A and 14B show that NK cells contribute to the therapeuticeffect of MICA-ferritin vaccine in B16F10 subcutaneous melanoma model.In FIG. 14A, 7 week old C57BL/6 female mice were immunized withMICA-ferritin vaccine (n=16) and boosted on day 14. The mice werechallenged with subcutaneous injection of 0.5×10⁶ B16F10 cellsexpressing MICA on day 21 after initial vaccination. Mice receivedintravenous injection of 200 μg of anti-NK1.1 antibody (n=8) or isotypecontrol antibody (n=8) 2 days prior to tumor challenge and twice a weekthereafter at a dose of 100 μg per mouse until the study endpoint. Tumorvolume was measured every other day. The mice were euthanized when thetumors reached ≥250 mm². Tumors reached their maximum volume by day 14in naïve, untreated, non-depleted control group (filled circle). NK celldepletion accelerated tumor growth in MICA-vaccinated group (emptytriangle) compared to MICA-vaccinated group that received isotypeantibody (filled square). In FIG. 14B, survival analysis of NK celldepletion experiment showing age matched naïve, untreated, non-depletedcontrol group in thick solid line; MICA-ferritin vaccinated, NK celldepleted in dashed line and MICA-ferritin vaccinated, isotype antibodyinjected mice in thin solid line.

FIGS. 15A and 15B show that serum polyclonal antibodies generated inresponse to MICA-ferritin vaccine prevent pulmonary metastasis ofB16F10-MICA tumor cells. In FIG. 15A, 8 week old Ighmtm1Cgn/J femalemice (n=12) were challenged with intravenous injection of 0.5×10⁶ MICAexpressing B16F10 melanoma cells. The mice were randomized into 3cohorts with 4 mice each. On days 1, 2, 4 and 6 after tumor challenge,the mice were injected (intraperitoneal route) with 100 μl of end pointsera from naïve, OVA-protein or MICA-ferritin immunized C57BL/6 mice.Mice were euthanized 14 days after tumor challenge; lungs were harvestedand fixed in 10% neutral-buffered formalin and the number of pulmonarymetastases was quantified. Mice injected with sera from MICA-ferritinvaccinated group (empty square) had significantly fewer lung metastasescompared to mice injected with sera from untreated, age matched controlgroup (filled circle) and OVA-protein immunized group. In FIG. 15B,sMICA level was lower in mice receiving sera from MICA-ferritinvaccinated group (empty square) compared to mice receiving sera fromnaïve or OVA-protein immunized group.

FIGS. 16A and 16B show that MICA-ferritin vaccine also controlsB16F10-MICB005 subcutaneous tumor growth. In FIG. 16A, 7 week oldC57BL/6 female mice (n=4) were immunized with MICA-ferritin vaccine andboosted on day 14. The mice were challenged with subcutaneous injectionof 0.5×10⁶B16F10 cells expressing MICB on day 21 after initialvaccination and the tumor volume was measured every other day.B16F10-MICB tumor growth in the MICA-ferritin immunized group was foundto be significantly slower (empty square) compared to the OVA-proteinimmunized control group (filled circle). In FIG. 16B, sMICB levels werenearly undetectable in sera of mice immunized with MICA-ferritin vaccine(empty square) while high levels of sMICB were detected within two weeksafter tumor challenge in the sera of the OVA-protein immunized controlgroup (filled circle).

FIG. 17 is a series of graphs showing the staining of B16 cell linesthat express MICA with sera from mice immunized with MICA-ferritinvaccine formulated with mesoporous silica rods (MSR) (dashed line) ordirect conjugation of CpG to MICA-ferritin (without MSR (thin solidline). These data illustrate that vaccination with CpG directlyconjugated to MICA-ferritin peptide induces a stronger immune responseto the MICA alpha 3 domain than the vaccine formulated with MSRscaffold. For MSR vaccine, 5 mg MSR+200 ug protein+100 ug CpG+1 ugGM-CSF, immunize on day 0; boost on day 14; serum from day 28. Fordirect conjugation, 200 ug protein conjugated to ˜5 ug CpG (primaryimmunization); boost (100 ug protein conjugated to ˜5 ug CpG+addavax(100 ul)+GM-CSF (1 ug); immunize on day 0; boost on day 21; serum fromday 28.

FIG. 18A is an electron micrograph of purified MICA α3—ferritinnanoparticles.

FIG. 18B is a picture of SDS-PAGE of vaccine protein following affinityand gel filtration chromatography.

FIG. 19 is a graph showing that polyclonal antibodies induced by MICA α3domain vaccine inhibit MICA shedding by human tumor cells. MICA sheddingby the human A375 melanoma cell line was quantified using a sandwichELISA. Addition of small quantities of sera (1-10 μl) from micevaccinated with MICA α3—ferritin strongly inhibited shedding whileaddition of sera from control mice had little effect.

FIGS. 20A-20C are a series of graphs showing that MICA-ferritin vaccineinduces secondary T cell responses to neoantigens. We examined whetherthe MICB α3 domain vaccine induces secondary responses to tumorneoantigens. Lymph node T cells were labeled with CFSE and cultured forthree days with four different neoantigen peptides previously identifiedfor B16F10 tumors as CD4 T cell epitopes. CD4 T cell responses wereidentified for three of the four peptides based on intracellular IFNγstaining in proliferating cells (CFSE^(low)). We hypothesize that MICAantibodies trigger Fc receptor mediated uptake of apoptotic tumorfragments by dendritic cells and thereby promote T cell responses toneoantigens. In FIGS. 20A-20B, B6 mice were immunized with MICBα3—ferritin or OVA (n=5/group) and injected with B16F10-MICB tumorcells. T cells were isolated from tumor-draining lymph nodes 10 daysafter tumor implantation and labeled with CF SE. T cells were culturedfor 3 days with CD11c+ spleen cells in the presence of four differentCD4 neoantigen peptides (10 μg/ml) previously identified for B16F10tumors. Intracellular IFNγ staining was performed and proliferating Tcells (CF SE-low) positive for intracellular IFNγ were quantified. Tcell responses to neoantigen peptides were compared between miceimmunized with the OVA control antigen (FIG. 20A) or MICB α3 domain(FIG. 20B). Both T cell populations were incubated in vitro with the M30neoantigen. In FIG. 20C, summary of T cell responses to threeneoantigens (M30, M44 and M48) for which enhanced T cell responses wereobserved in MICB immunized mice.

FIGS. 21A-21B are graphs showing immunization with MICA-ferritinnanoparticles conjugated with CpG induces high-titer antibodies. In FIG.21A, macaque MICAS-ferritin was conjugated to CpG ODN 1826 by CLICKchemistry (protein-oligo conjugation kit, Solulink). Briefly, S-HyNic(succinimidyl-6-hydrazino-nicotinamide) linker was conjugated to theprotein through primary amines on the lysine and S-4B(succinimidyl-4-formylbenzamide) linker was added to CpG oligo. Themodified protein and oligo were incubated in a catalyzed conjugationreaction. Following this reaction, excess of unconjugated CpG wasremoved by size exclusion chromatography. Protein-oligo conjugate bond(stable, bis-arylhydrazone bond) formed is UV traceable at 350 nm (seegraph). In FIG. 21B, the CpG conjugated protein was used to immunizeC57BL/6 mice. MICAS specific antibodies in the serum were analyzed onday 14 by labeling of B16-MICA cells. The CpG linked protein inducedhigher titer antibodies (thin solid line) compared to the MICA-ferritinprotein formulated with the scaffold (dashed line; thick solid linedemonstrates background staining levels).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a vaccine for cancer. More specifically,the present invention provides a MIC alpha 3-domain vaccine that canelicit an immune response against MIC alpha 3-domain. Importantly, thevaccine elicits antibodies against the MIC α3 domain, but not againstthe α1-α2 domains of MIC as not to interfere with the binding of theα1-α2 domains to the NKG2D receptor on NK cells.

The purpose of the vaccine is to induce polyclonal antibodies that bindto the membrane-proximal Ig domain of MICA and inhibit proteolyticshedding of this protein from tumor cells. The MICA alpha 3 domain wasexpressed on the surface of nanoparticles. Specifically, the MICA alpha3 domain coding sequence was fused to the ferritin sequence (from H.pylori), given that ferritin spontaneously forms nanoparticles. Thevaccine was formulated either with an immunization scaffold (mesoporoussilica rods) using CpG as the adjuvant and GM-CSF to recruit dendriticcells to the injection site or directly conjugating CpG to the MICAalpha 3 domain-ferritin fusion protein and GM-CSF. It was found thatinjections of these vaccines induced high-titer antibodies directedagainst the MICA alpha 3 domain. Surprisingly, the MICA alpha 3domain-ferritin fusion protein that had CpG directly conjugated achievedhigher antibody titers.

These antibodies induced by the vaccine composition of the inventionbound to multiple MICA alleles and stained tumor cells that were MICApositive. Importantly, these polyclonal antibodies inhibited shedding ofMICA by tumor cells. The in vivo efficacy of the vaccine was tested in ametastatic mouse model of melanoma. B 16F10 melanoma cells weregenetically modified to express MICA and injected intravenously afterthe mice had been vaccinated twice. The vaccine provided a high level ofprotection while control mice had large numbers of pulmonary metastases(˜150-200).

The vaccine of the invention is conceptually different from conventionalcancer vaccines that attempt to induce an immune response thateliminates all cancer cells expressing a particular antigen. In contrastthe purpose of the vaccine of the invention is to prevent tumor escapefrom an important immune surveillance pathway. This vaccine will be safebased on the study of patients with MICA antibodies and the fact thatMIC expression flags cells for elimination by cytotoxic lymphocytes. Thebenefits of the vaccine approach are: low cost of a vaccine, long-termprotection against escape from immune surveillance, induction ofpolyclonal antibodies that inhibit shedding and rapidly clear shed MICby formation of immune complexes and induction of a T cell responseagainst other tumor antigens by enhanced uptake of apoptotic tumorfragments by dendritic cells.

Also provided by the invention are self-assembling ferritin-based,nanoparticles that display immunogenic portions of MICA alpha 3 domainon their surface. Optionally, the nanoparticles further include a CpGoligonucleotide. For example, CpG oligonucleotide is covalently coupledto the MICA alpha 3 domain-ferritin fusion protein. Such nanoparticlesare useful for vaccinating individuals. Accordingly, the presentinvention also relates to fusion proteins for producing suchnanoparticles and nucleic acid molecules encoding such proteins.Additionally, the present invention relates to, methods of producingnanoparticles of the present invention, and methods of using suchnanoparticles to vaccinate individuals.

Also provided by the invention are vaccine compositions comprising a MICalpha 3-domain peptide joined to a CpG oligonucleotide.

Vaccines Against Mic Alphas Domain Protein

The invention provides a vaccine composition suitable for administrationto a human comprising, as an immunogenic component, at least one MICalpha 3-domain peptide. The MIC alpha 3-domain peptide comprises orconsists of the full-length alpha 3 domain of MICA or MICB, which domaincorresponds to amino acids 181 to 274 of SEQ ID NO: 1 or SEQ ID NO: 2.Optionally, the peptide includes one or more flanking amino acids. Inthis context, the term “flanking amino acids” refers to the amino acidsadjacent to the MIC alpha 3-domain sequence in the full-length referencesequence [SEQ ID NO: 1 for MICA or SEQ ID NOs: 2 for MICB]. In certainembodiments, the peptide comprises 2, 4, 6, 8, or 10 flanking aminoacids on either its N- or C-terminal end, or both. In some embodimentsthe vaccine peptide is non glycosylated.

Amino Acid Sequence of MICA HSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRYDRQKCRAKP QGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGL HSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNVETEEWTVPQS SRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLES SVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNITLTWRQD GVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYME HSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAAAAAIFVIIIFYV RCCKKKTSAAEGPELVSLQVLDQHPVGTSDHRDATQLGFQPLMS  ALGSTGSTEGA (SEQ ID NO: 1)Amino Acid Sequence of MICB  PHSLRYNLMVLSQDGSVQSGFLAEGHLDGQPFLRYDRQKRRA KPQGQWAEDVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKG GLHSLQEIRVCEIHEDSSTRGSRHFYYDGELFLSQNLETQESTVP QSSRAQTLAIVINVTNFWKEDAMKTKTHYRAMQADCLQKLQRY LKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTW RQDGVSLSHNTQQWGDVLPDGGTYQTWVATRIRQGEEQRFTCY MEHSGNHGTHPVPSGKALVLQSQRTDFPYVSAAMPCFVIIIILCVP CCKKKTSAAEGPELVSLQVLDQHPVGTGDHRDAAQLGFQPLMSA  TGSTGSTEGA (SEQ ID NO: 2)

In a preferred embodiment, the vaccine comprises a peptide having theamino acid sequence of:

(SEQ ID NO: 3) RTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRIS QGEEQRFTCYMEHSGNHSTHPVPSGK VLVLQSHWQTFH  or (SEQ ID NO: 4) RTVPPMVQVTRSEASEGQITVTCRASGFYPWNINLSWRQDGVSLSHDTQQWGDVLPD GNGTYQTWVA TRISQGEEQRFTCYMEHSGQHSTHPVPSG KVLVLQSHWQTFH. 

In another embodiment, the vaccine composition comprises a nucleic acidencoding the MIC alpha 3-domain sequence. The nucleic acid may be in theform of an expression vector, for example a plasmid or a viral vector,or the nucleic acid may be packaged into nanoparticles. In oneembodiment, the nucleic acid is delivered to a subject by injection. Inone embodiment, the nucleic acid is injected as purified DNA or in theform of nanoparticles. In one embodiment, modified immune cells whichhave been modified to express the nucleic acid are injected. In oneembodiment, the immune cells are modified via transfection or infectionin vitro with a vector comprising the nucleic acid.

The peptides which form or are incorporated into the vaccinecompositions of the invention are preferably purified from contaminatingchemical precursors, if chemically synthesized, or substantially free ofcellular material from the cell or tissue source from which they arederived. In a specific embodiment, the peptides are 60%, preferably 65%,70%, 75%, 80%, 85%, 90%, 95%, or 99% free of contaminating chemicalprecursors, proteins, lipids or nucleic acids. In a preferredembodiment, the peptides are substantially free of contaminating virus.Preferably, each composition for administering to a subject is at least95%, at least 97%, or at least 99% free of contaminating virus.

In one embodiment, the MIC alpha 3-domain peptide of a vaccinecomposition of the invention comprises or consists of one or morepeptides that is at least 90%, at least 95%, at least 98%, or at least99% identical to a peptide including amino acids 181 to 274 of SEQ IDNO: 1 or SEQ ID NO: 2. In this context, the term “similar” refers toamino acid sequence similarity which is defined according to the numberof conservative and non-conservative amino acid changes in a querysequence relative to a reference sequence. Conservative andnon-conservative amino acid changes are known in the art. See, forexample, W. R. Taylor, The Classification of Amino Acid Conservation, J.Theor. Biol. 1986 119:205-218, and D. Bordo and P. Argos, Suggestionsfor “Safe” Residue Substitutions in Site-Directed Mutagensis, 1991 J.Mol. Biol. 217:721-729. Generally, a conservative amino acid changerefers to a substitution of one amino acid for another amino acid havingsubstantially similar chemical properties, specifically with referenceto the amino acid side chains. A non-conservative change refers to asubstitution of one amino acid for another amino acid havingsubstantially different chemical properties. Generally, conservativesubstitutions are those recognized in the art as being unlikely toaffect the overall structure or biological function of the polypeptide,while non-conservative changes are recognized as more likely to affectstructure and function.

Non-limiting examples of a conservative amino acid change includesubstitution of amino acids within the following groups: aliphatic,aromatic, polar, nonpolar, acidic, basic, phosphorylatable hydrophobic,hydrophilic, small nonpolar, small polar, large nonpolar, and largepolar. Non-limiting examples of non-conservative amino acid changesinclude substitutions of amino acids between the foregoing groups.

In one embodiment, a conservative amino acid change is a substitution inwhich the substitution matrix for the pair of residues has a positivevalue. Examples of amino acid substitution matrices are known in theart, for example the BLOSUM50 matrix or the PAM250 matrix (see W. A.Pearson, Rapid and Sensitive Sequence Comparison with FASTP and FASTA,Meth. Enzymology, 1990 183:63-98, ed. R. Doolittle, Academic Press, SanDiego). For further examples of scoring matrices and a comparisonbetween them see M. S. Johnson and J. P. Overington, 1993, A StructuralBasis for Sequence Comparisons: An Evaluation of Scoring Methodologies,J. Mol. Biol. 233:716-738.

In a preferred embodiment, a conservative amino acid change is asubstitution of one amino acid for another amino acid within the samechemical group wherein the groups are selected from neutral and polaramino acids (Ser, Thr, Pro, Ala, Gly, Asn, Gln), negatively charged andpolar amino acids (Asp, Glu), positively charged and polar amino acids(His, Arg, Lys), nonpolar amino acids lacking a ring structure (Met,Ile, Leu, Val), nonpolar amino acids having a ring structure (Phe, Tyr,Trp), and Cysteine.

In various embodiments, the peptide is conjugated to a CpGoligonucleotide sequence.

In other embodiments, the peptide is conjugated to a carrier protein.The term “carrier protein” is intended to cover both small peptides andlarge polypeptides (>10 kDa). The carrier protein may be any peptide orprotein. It may comprise one or more T-helper epitopes. The carrierprotein may be tetanus toxoid (TT), tetanus toxoid fragment C, non-toxicmutants of tetanus toxin [note all such variants of TT are considered tobe the same type of carrier protein for the purposes of this invention],polypeptides comprising tetanus toxin T-cell epitopes such as N19(WO2006/067632), diphtheria toxoid (DT), CRM197, other non-toxic mutantsof diphtheria toxin such as CRM176, CRM 197, CRM228, CRM 45 (Uchida etal J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103and CRM107 and other mutations described by Nicholls and Youle inGenetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992;deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. No. 4,709,017 or 4,950,740;mutation of at least one or more residues Lys 516, Lys 526, Phe 530and/or Lys 534 and other mutations disclosed in U.S. Pat. No. 5,917,017or 6,455,673; or fragment disclosed in U.S. Pat. No. 5,843,711] (noteall such variants of DT are considered to be the same type of carrierprotein for the purposes of this invention), pneumococcal pneumolysin(Kuo et al (1995) Infect Immun 63; 2706-13), OMPC (meningococcal outermembrane protein—usually extracted from N. meningitidissero groupB—EP0372501), synthetic peptides (EP0378881, EP0427347), heat shockproteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668,EP0471177), cytokines, lymphokines, growth factors or hormones (WO91/01146), artificial proteins comprising multiple human CD4+ T cellepitopes from various pathogen derived antigens (Falugi et al (2001) EurJ Immunol 31; 3816-3824) such as N19 protein (Baraldoi et al (2004)Infect Immun 72; 4884-7) pneumococcal surface protein PspA (WO02/091998), iron uptake proteins (WO 01/72337), toxin A or B of C.difficile (WO 00/61761), H. influenzae Protein D (EP594610 and WO00/56360), pneumococcal PhtA (WO 98/18930, also referred to Sp36),pneumococcal PhtD (disclosed in WO 00/37105, and is also referred toSp036D), pneumococcal PhtB (disclosed in WO 00/37105, and is alsoreferred to Sp036B), or PhtE (disclosed in WO00/30299 and is referred toas BVH-3).

In one embodiment, the carrier protein can be selected from the groupconsisting of: tetanus toxoid (TT), fragment C of tetanus toxoid,diphtheria toxoid (DT), CRM197, Pneumolysin (Ply), protein D, PhtD,PhtDE and N19. In one embodiment the carrier protein is CRM197.

Vaccines Comprising Ha-Ferritin Fusion Proteins

The inventors have also discovered that fusion of a MIC alpha 3-domainpeptide with ferritin protein a MIC alpha 3-ferritin fusion protein)results in a vaccine that elicits a robust immune response to cancer.Such MIC alpha 3-ferritin fusion proteins self-assemble intonanoparticles that display immunogenic portions of the MIC alpha3-domain peptide on their surface. These nanoparticles are useful forvaccinating individuals against MIC alpha 3-domain. Thus, one embodimentof the present invention is an MIC alpha 3-ferritin fusion proteincomprising a monomeric ferritin subunit disclosed herein joined to a MICalpha 3-domain peptide disclosed herein. The MIC alpha 3-ferritin fusionprotein is capable of self-assembling into nanoparticles. In variousaspects the fusion protein further comprises a CpG oligonucleotidesequence. The CpG oligonucleotide sequence can be covalently attached tothe MIC alpha 3-ferritin fusion protein.

Ferritin is a globular protein found in all animals, bacteria, andplants, that acts primarily to control the rate and location ofpolynuclear Fe(III)₂O₃ formation through the transportation of hydratediron ions and protons to and from a mineralized core. The globular formof ferritin is made up of monomeric subunit proteins (also referred toas monomeric ferritin subunits), which are polypeptides having amolecule weight of approximately 17-20 kDa. Each monomeric ferritinsubunit has the topology of a helix bundle which includes a fourantiparallel helix motif, with a fifth shorter helix (the c-terminalhelix) lying roughly perpendicular to the long axis of the 4 helixbundle. According to convention, the helices are labeled ‘A, B, C, and D& E’ from the N-terminus respectively. The N-terminal sequence liesadjacent to the capsid three-fold axis and extends to the surface, whilethe E helices pack together at the four-fold axis with the C-terminusextending into the particle core. The consequence of this packingcreates two pores on the capsid surface. It is expected that one or bothof these pores represent the point by which the hydrated iron diffusesinto and out of the capsid. Following production, these monomericferritin subunit proteins self-assemble into the globular ferritinprotein. Thus, the globular form of ferritin comprises 24 monomeric,ferritin subunit proteins, and has a capsid-like structure having 432symmetry.

According to the present invention, a monomeric ferritin subunit of thepresent invention is a full length, single polypeptide of a ferritinprotein, or any portion thereof, which is capable of directingself-assembly of monomeric ferritin subunits into the globular form ofthe protein. Amino acid sequences from monomeric ferritin subunits ofany known ferritin protein can be used to produce fusion proteins of thepresent invention, so long as the monomeric ferritin subunit is capableof self-assembling into a nanoparticle displaying MIC alpha 3-domain onits surface. In one embodiment, the monomeric subunit is from a ferritinprotein selected from the group consisting of a bacterial ferritinprotein, a plant ferritin protein, an algal ferritin protein, an insectferritin protein, a fungal ferritin protein and a mammalian ferritinprotein. In one embodiment, the ferritin protein is from Helicobacterpylori.

MIC alpha 3-ferritin fusion proteins of the present invention need notcomprise the full-length sequence of a monomeric subunit polypeptide ofa ferritin protein. Portions, or regions, of the monomeric ferritinsubunit protein can be utilized so long as the portion comprises anamino acid sequence that directs self-assembly of monomeric ferritinsubunits into the globular form of the protein. One example of such aregion is located between amino acids 5 and 167 of the Helicobacterpylori ferritin protein. More specific regions are described in Zhang,Y. Self-Assembly in the Ferritin Nano-Cage Protein Super Family. 2011,Int. J. Mol. Sci., 12, 5406-5421, which is incorporated herein byreference in its entirety.

One embodiment of the present invention is an MIC alpha 3-ferritinfusion protein comprising an MIC alpha 3-domain protein of the presentinvention joined to at least 25 contiguous amino acids, at least 50contiguous amino acids, at least 75 contiguous amino acids, at least 100contiguous amino acids, or at least 150 contiguous amino acids from amonomeric ferritin subunit, wherein the MIC alpha 3-ferritin fusionprotein is capable of self-assembling into nanoparticles. One embodimentof the present invention is an MIC alpha 3-ferritin fusion proteincomprising an MIC alpha 3-domain protein of the present invention joinedto at least 25 contiguous amino acids, at least 50 contiguous aminoacids, at least 75 contiguous amino acids, at least 100 contiguous aminoacids, or at least 150 contiguous amino acids from the region of aferritin protein corresponding to the amino acid sequences of theHelicobacter pylori ferritin monomeric subunit that direct self-assemblyof the monomeric subunits into the globular form of the ferritinprotein, wherein the MIC alpha 3-ferritin fusion protein is capable ofself-assembling into nanoparticles.

It is well-known in the art that some variations can be made in theamino acid sequence of a protein without affecting the activity of theprotein. Such variations include insertion of amino acid residues,deletions of amino acid residues, and substitutions of amino acidresidues. Thus, in one embodiment, the sequence of the monomericferritin subunit is divergent enough from the sequence of a ferritinsubunit naturally found in a mammal, such that when the variantmonomeric ferritin subunit is introduced into the mammal, it does notresult in the production of antibodies that react with the mammal'snatural ferritin protein. According to the present invention, such amonomeric subunit is referred to as immunogenically neutral. Oneembodiment of the present invention is an MIC alpha 3-ferritin fusionprotein comprising an MIC alpha 3-domain protein of the presentinvention joined to an amino acid sequence at least 80%, at least 85%,at least 90%, at least 95%, and at least 97% identical to the amino acidsequence of a monomeric ferritin subunit that is responsible fordirecting self-assembly of the monomeric ferritin subunits into theglobular form of the protein, wherein the MIC alpha 3-ferritin fusionprotein is capable of self-assembling into nanoparticles. In oneembodiment, the HA-ferritin fusion protein comprises a polypeptidesequence identical in sequence to a monomeric ferritin subunit. Oneembodiment of the present invention is an MIC alpha 3-ferritin fusionprotein comprising an MIC alpha 3-domain protein of the presentinvention joined to an amino acid sequence at least 80%, at least 85%,at least 90%, at least 95%, and at least 97% identical to the amino acidsequence of a monomeric ferritin subunit from Helicobacter pylori,wherein the MIC alpha 3-ferritin fusion protein is capable ofself-assembling into nanoparticles.

In some embodiments, it may be useful to engineer mutations into theamino acid sequences of proteins of the present invention. For example,it may be useful to alter sites such as enzyme recognition sites orglycosylation sites in the monomeric ferritin subunit, the trimerizationdomain, or linker sequences, in order to give the fusion proteinbeneficial properties (e.g., solubility, half-life, mask portions of theprotein from immune surveillance). In this regard, it is known that themonomeric subunit of ferritin is not glycosylated naturally. However, itcan be glycosylated if it is expressed as a secreted protein inmammalian or yeast cells. Thus, in one embodiment, potential N-linkedglycosylation sites in the amino acid sequences from the monomericferritin subunit are mutated so that the mutated ferritin subunitsequences are no longer glycosylated at the mutated site.

Proteins of the present invention are encoded by nucleic acid moleculesof the present invention. In addition, they are expressed by nucleicacid constructs of the present invention. As used herein a nucleic acidconstruct is a recombinant expression vector, i.e., a vector linked to anucleic acid molecule encoding a protein such that the nucleic acidmolecule can effect expression of the protein when the nucleic acidconstruct is administered to, for example, a subject or an organ, tissueor cell. The vector also enables transport of the nucleic acid moleculeto a cell within an environment, such as, but not limited to, anorganism, tissue, or cell culture. A nucleic acid construct of thepresent disclosure is produced by human intervention. The nucleic acidconstruct can be DNA, RNA or variants thereof. The vector can be a DNAplasmid, a viral vector, or other vector. In one embodiment, a vectorcan be a cytomegalovirus (CMV), retrovirus, adenovirus, adeno-associatedvirus, herpes virus, vaccinia virus, poliovirus, sindbis virus, or anyother DNA or RNA virus vector. In one embodiment, a vector can be apseudotyped lentiviral or retroviral vector. In one embodiment, a vectorcan be a DNA plasmid. In one embodiment, a vector can be a DNA plasmidcomprising viral components and plasmid components to enable nucleicacid molecule delivery and expression. Methods for the construction ofnucleic acid constructs of the present disclosure are well known. See,for example, Molecular Cloning: a Laboratory Manual, 3.sup.rd edition,Sambrook et al. 2001 Cold Spring Harbor Laboratory Press, and CurrentProtocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons,1994. In one embodiment, the vector is a DNA plasmid, such as a CMV/Rplasmid such as CMV/R or CMV/R 8 KB (also referred to herein as CMV/R 8kb). Examples of CMV/R and CMV/R 8 kb are provided herein. CMV/R is alsodescribed in U.S. Pat. No. 7,094,598 B2, issued Aug. 22, 2006.

As used herein, a nucleic acid molecule comprises a nucleic acidsequence that encodes MIC alpha 3-domain peptide immunogen, a ferritinmonomeric subunit, and/or an MIC alpha 3-ferritin fusion protein of thepresent invention. A nucleic acid molecule can be producedrecombinantly, synthetically, or by a combination of recombinant andsynthetic procedures. A nucleic acid molecule of the disclosure can havea wild-type nucleic acid sequence or a codon-modified nucleic acidsequence to, for example, incorporate codons better recognized by thehuman translation system. In one embodiment, a nucleic acid molecule canbe genetically-engineered to introduce, or eliminate, codons encodingdifferent amino acids, such as to introduce codons that encode anN-linked glycosylation site. Methods to produce nucleic acid moleculesof the disclosure are known in the art, particularly once the nucleicacid sequence is known. It is to be appreciated that a nucleic acidconstruct can comprise one nucleic acid molecule or more than onenucleic acid molecule. It is also to be appreciated that a nucleic acidmolecule can encode one protein or more than one protein.

In one embodiment, the monomeric subunit of ferritin is from theferritin protein of Helicobacter pylori.

Also embodied in the present invention are nucleic acid sequences thatare variants of nucleic acid sequence encoding protein of the presentinvention. Such variants include nucleotide insertions, deletions, andsubstitutions, so long as they do not affect the ability of fusionproteins of the present invention to self-assemble into nanoparticles,or significantly affect the ability of the MIC alpha 3-domain portion offusion proteins to elicit an immune response to MIC alpha 3-domainprotein.

Also encompassed by the present invention are expression systems forproducing fusion proteins of the present invention. In one embodiment,nucleic acid molecules of the present invention are operationally linkedto a promoter. As used herein, operationally linked means that proteinsencoded by the linked nucleic acid molecules can be expressed when thelinked promoter is activated. Promoters useful for practicing thepresent invention are known to those skilled in the art. One embodimentof the present invention is a recombinant cell comprising a nucleic acidmolecule of the present invention. One embodiment of the presentinvention is a recombinant virus comprising a nucleic acid molecule ofthe present invention.

As indicated above, the recombinant production of the ferritin fusionproteins of the present invention can take place using any suitableconventional recombinant technology currently known in the field. Forexample, molecular cloning a fusion protein, such as ferritin with asuitable protein such as the recombinant MIC alpha 3-domain protein, canbe carried out via expression in E. coli with the suitable monomericsubunit protein, such as the Helicobacter pylori ferritin monomericsubunit. The construct may then be transformed into protein expressioncells, grown to suitable size, and induced to produce the fusionprotein.

As has been described, because MIC alpha 3-ferritin fusion proteins ofthe present invention comprise a monomeric subunit of ferritin, they canself-assemble. According to the present invention, the supramoleculeresulting from such self-assembly is referred to as a MIC alpha 3expressing ferritin based nanoparticle. For ease of discussion, the MICalpha 3 expressing ferritin based nanoparticle will simply be referredto as a, or the, nanoparticle (np). Nanoparticles of the presentinvention have the same structural characteristics as the ferritinproteins described earlier. That is, they contain 24 subunits and have432 symmetry. In the case of nanoparticles of the present invention, thesubunits are the fusion proteins comprising a ferritin monomeric subunitjoined to an MIC alpha 3-domain protein. Such nanoparticles display atleast a portion of the MIC alpha 3-domain protein on their surface.Thus, one embodiment of the present invention is a nanoparticlecomprising an MIC alpha 3-ferritin fusion protein, wherein the fusionprotein comprises a monomeric ferritin subunit joined to a MIC alpha3-domain protein. In one embodiment, the nanoparticle is an octahedron.

Because MIC alpha 3-ferritin fusion proteins and nanoparticles of thepresent invention can elicit an immune response to an MIC alpha 3-domainprotein, they can be used as vaccines to treat cancer. According to thepresent invention a vaccine can be a MIC alpha 3-domain peptideimmunogen, an MIC alpha 3-ferritin fusion protein, or a nanoparticle ofthe present invention. Vaccines of the present invention can alsocontain other components such as adjuvants, buffers and the like.Although any adjuvant can be used, preferred embodiments can contain:chemical adjuvants such as aluminum phosphate, benzyalkonium chloride,ubenimex, and QS21; genetic adjuvants such as the IL-2 gene or fragmentsthereof, the granulocyte macrophage colony-stimulating factor (GM-CSF)gene or fragments thereof, the IL-18 gene or fragments thereof, thechemokine (C-C motif) ligand 21 (CCL21) gene or fragments thereof, theIL-6 gene or fragments thereof, CpG, LPS, TLR agonists, and other immunestimulatory genes; protein adjuvants such IL-2 or fragments thereof, thegranulocyte macrophage colony-stimulating factor (GM-CSF) or fragmentsthereof, IL-18 or fragments thereof, the chemokine (C-C motif) ligand 21(CCL21) or fragments thereof, IL-6 or fragments thereof, CpG, LPS, TLRagonists and other immune stimulatory cytokines or fragments thereof;lipid adjuvants such as cationic liposomes, N3 (cationic lipid),monophosphoryl lipid A (MPL1); other adjuvants including cholera toxin,enterotoxin, Fms-like tyrosine kinase-3 ligand (Flt-3L), bupivacaine,marcaine, and levamisole.

Mesoporous Silica

The vaccine composition according to the invention can further comprisean immunization scaffold. In one embodiment, the immunization scaffoldis mesoporous silica nanoparticles (MSR). MSR can be in any shape orform, such as rods, spheres, wires, cubes, or polyhedrons. The shape orform of MSR is typically the result of specific reaction conditions. Forexample, mesoporous silica nanoparticles can be synthesized by anymethod known in the art, such as reacting tetraethyl orthosilicate witha template made of micellar rods. The result is a collection ofnano-sized spheres or rods that are filled with a regular arrangement ofpores. The template can then be removed by washing with a solventadjusted to the proper pH. In another technique, the mesoporous particlecould be synthesized using a simple sol-gel method or a spray dryingmethod. Tetraethyl orthosilicate is also used with an additional polymermonomer (as a template). Other methods include those described in U.S.Patent Publication 20150072009, 20120264599 and 20120256336, herebyincorporated by reference.

Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a proteinsecreted by macrophages, T cells, mast cells, endothelial cells andfibroblasts. Specifically, GM-CSF is a cytokine that functions as awhite blood cell growth factor. GM-CSF stimulates stem cells to producegranulocytes and monocytes. Monocytes exit the blood stream, migrateinto tissue, and subsequently mature into macrophages.

Scaffold devices described herein comprise and release GM-CSFpolypeptides to attract host DCs to the device. Contemplated GM-CSFpolypeptides are isolated from endogenous sources or synthesized in vivoor in vitro. Endogenous GM-CSF polypeptides are isolated from healthyhuman tissue. Synthetic GM-CSF polypeptides are synthesized in vivofollowing transfection or transformation of template DNA into a hostorganism or cell, e.g. a mammal or cultured human cell line.Alternatively, synthetic GM-CSF polypeptides are synthesized in vitro bypolymerase chain reaction (PCR) or other art-recognized methods (e.g.,Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3(1989), herein incorporated by reference).

GM-CSF polypeptides are modified to increase protein stability in vivo.Alternatively, GM-CSF polypeptides are engineered to be more or lessimmunogenic. Endogenous mature human GM-CSF polypeptides areglycosylated, reportedly, at amino acid residues 23 (leucine), 27(asparagine), and 39 (glutamic acid) (see U.S. Pat. No. 5,073,627).GM-CSF polypeptides of the present invention are modified at one or moreof these amino acid residues with respect to glycosylation state.

GM-CSF polypeptides are recombinant. Alternatively, GM-CSF polypeptidesare humanized derivatives of mammalian GM-CSF polypeptides. Exemplarymammalian species from which GM-CSF polypeptides are derived include,but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat,dog, monkey, or primate. In a preferred embodiment, GM-CSF is arecombinant human protein (PeproTech, Catalog #300-03). Alternatively,GM-CSF is a recombinant murine (mouse) protein (PeproTech, Catalog#315-03). Finally, GM-CSF is a humanized derivative of a recombinantmouse protein.

Human Recombinant GM-CSF (PeproTech, Catalog #300-03) is encoded by the following polypeptide sequence:  (SEQ ID NO: 26)MAPARSPSPS TQPWEHVNAI QEARRLLNLS RDTAAEMNET VEVISEMFDL QEPTCLQTRL ELYKQGLRGS LTKLKGPLTM MASHYKQHCP PTPETSCATQ IITFESFKEN LKDFLLVIPF DCWEPVQE Murine Recombinant GM-CSF (PeproTech, Catalog #315-03) is encoded by thefollowing polypeptide sequence:  (SEQ ID NO: 27)MAPTRSPITV TRPWKHVEAI KEALNLLDDM PVTLNEEVEV VSNEFSFKKL TCVQTRLKIF EQGLRGNFTK LKGALNMTAS YYQTYCPPTP ETDCETQVTT YADFIDSLKT FLTDIPFECK KPVQK Human Endogenous GM-CSF is encoded by the following mRNA sequence (NCBI Accession No. NM 000758 and SEQ ID NO: 28):  (SEQ ID NO: 28)acacagagag aaaggctaaa gttctctgga ggatgtggct gcagagcctg ctgctcttgg  61 gcactgtggc ctgcagcatc tctgcacccg cccgctcgcc cagccccagc acgcagccct 121 gggagcatgt gaatgccatc caggaggccc ggcgtctcct gaacctgagt agagacactg 181 ctgctgagat gaatgaaaca gtagaagtca tctcagaaat gtttgacctc caggagccga 241 cctgcctaca gacccgcctg gagctgtaca agcagggcct gcggggcagc ctcaccaagc 301 tcaagggccc cttgaccatg atggccagcc actacaagca gcactgccct ccaaccccgg 361 aaacttcctg tgcaacccag attatcacct ttgaaagttt caaagagaac ctgaaggact 421 ttctgcttgt catccccttt gactgctggg agccagtcca ggagtgagac cggccagatg 481 aggctggcca agccggggag ctgctctctc atgaaacaag agctagaaac tcaggatggt 541 catcttggag ggaccaaggg gtgggccaca gccatggtgg gagtggcctg gacctgccct 601 gggccacact gaccctgata caggcatggc agaagaatgg gaatatttta tactgacaga 661 aatcagtaat atttatatat ttatattttt aaaatattta tttatttatt tatttaagtt 721 catattccat atttattcaa gatgttttac cgtaataatt attattaaaa atatgcttct 781 aHuman Endogenous GM-CSF is encoded by the following amino acid sequence(NCBI Accession No. NP000749.2 and SEQ ID NO: 29):  (SEQ ID NO: 29)MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDT AAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMM ASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE 

Cytosine-Guanosine (CpG) Oligonucleotide (CpG-ODN) Sequences

CpG sites are regions of deoxyribonucleic acid (DNA) where a cysteinenucleotide occurs next to a guanine nucleotide in the linear sequence ofbases along its length (the “p” represents the phosphate linkage betweenthem and distinguishes them from a cytosine-guanine complementary basepairing). CpG sites play a pivotal role in DNA methylation, which is oneof several endogenous mechanisms cells use to silence gene expression.Methylation of CpG sites within promoter elements can lead to genesilencing. In the case of cancer, it is known that tumor suppressorgenes are often silenced while oncogenes, or cancer-inducing genes, areexpressed. CpG sites in the promoter regions of tumor suppressor genes(which prevent cancer formation) have been shown to be methylated whileCpG sites in the promoter regions of oncogenes are hypomethylated orunmethylated in certain cancers. The TLR-9 receptor binds unmethylatedCpG sites in DNA.

The vaccine composition described herein comprises CpG oligonucleotides.CpG oligonucleotides are isolated from endogenous sources or synthesizedin vivo or in vitro. Exemplary sources of endogenous CpGoligonucleotides include, but are not limited to, microorganisms,bacteria, fungi, protozoa, viruses, molds, or parasites. Alternatively,endogenous CpG oligonucleotides are isolated from mammalian benign ormalignant neoplastic tumors. Synthetic CpG oligonucleotides aresynthesized in vivo following transfection or transformation of templateDNA into a host organism. Alternatively, Synthetic CpG oligonucleotidesare synthesized in vitro by polymerase chain reaction (PCR) or otherart-recognized methods (Sambrook, J., Fritsch, E. F., and Maniatis, T.,Molecular Cloning: A Laboratory Manual. Cold Spring Harbor LaboratoryPress, NY, Vol. 1, 2, 3 (1989), herein incorporated by reference).

CpG oligonucleotides are presented for cellular uptake by dendriticcells. For example, naked CpG oligonucleotides are used. The term“naked” is used to describe an isolated endogenous or syntheticpolynucleotide (or oligonucleotide) that is free of additionalsubstituents. In another embodiment, CpG oligonucleotides are bound toone or more compounds to increase the efficiency of cellular uptake.Alternatively, or in addition, CpG oligonucleotides are bound to one ormore compounds to increase the stability of the oligonucleotide withinthe scaffold and/or dendritic cell. CpG oligonucleotides are optionallycondensed prior to cellular uptake. For example, CpG oligonucleotidesare condensed using polyethylimine (PEI), a cationic polymer thatincreases the efficiency of cellular uptake into dendritic cells.

CpG oligonucleotides can be divided into multiple classes. For example,exemplary CpG-ODNs encompassed by compositions, methods and devices ofthe present invention are stimulatory, neutral, or suppressive. The term“stimulatory” describes a class of CpG-ODN sequences that activate TLR9.The term “neutral” describes a class of CpG-ODN sequences that do notactivate TLR9. The term “suppressive” describes a class of CpG-ODNsequences that inhibit TLR9. The term “activate TLR9” describes aprocess by which TLR9 initiates intracellular signaling.

Stimulatory CpG-ODNs can further be divided into three types A, B and C,which differ in their immune-stimulatory activities. Type A stimulatoryCpG ODNs are characterized by a phosphodiester central CpG-containingpalindromic motif and a phosphorothioate 3′ poly-G string. Followingactivation of TLR9, these CpG ODNs induce high IFN-.alpha. productionfrom plasmacytoid dendritic cells (pDC). Type A CpG ODNs weaklystimulate TLR9-dependent NF-.kappa.B signaling.

Type B stimulatory CpG ODNs contain a full phosphorothioate backbonewith one or more CpG dinucleotides. Following TLR9 activation, theseCpG-ODNs strongly activate B cells. In contrast to Type A CpG-ODNs, TypeB CpG-ODNS weakly stimulate IFN-.alpha. secretion.

Type C stimulatory CpG ODNs comprise features of Types A and B. Type CCpG-ODNs contain a complete phosphorothioate backbone and a CpGcontaining palindromic motif. Similar to Type A CpG ODNs, Type C CpGODNs induce strong IFN-.alpha. production from pDC. Similar to Type BCpG ODNs, Type C CpG ODNs induce strong B cell stimulation.

Exemplary stimulatory CpG ODNs comprise, but are not limited to, ODN1585, ODN 1668, ODN 1826, ODN 2006, ODN 2006-G5, ODN 2216, ODN 2336, ODN2395, ODN M362 (all InvivoGen). The present invention also encompassesany humanized version of the preceding CpG ODNs. In one preferredembodiment, compositions, methods, and devices of the present inventioncomprise ODN 1826 (the sequence of which from 5′ to 3′ istccatgacgttcctgacgtt, wherein CpG elements are bolded, SEQ ID NO: 30).

Neutral, or control, CpG ODNs that do not stimulate TLR9 are encompassedby the present invention. These ODNs comprise the same sequence as theirstimulatory counterparts but contain GpC dinucleotides in place of CpGdinucleotides.

Exemplary neutral, or control, CpG ODNs encompassed by the presentinvention comprise, but are not limited to, ODN 1585 control, ODN 1668control, ODN 1826 control, ODN 2006 control, ODN 2216 control, ODN 2336control, ODN 2395 control, ODN M362 control (all InvivoGen). The presentinvention also encompasses any humanized version of the preceding CpGODNs.

Methods of Treating and Administration

The vaccine compositions of the present invention are useful for theprophylaxis and treatment of cancer. Accordingly, the present inventionprovides methods of prophylaxis against cancer in a subject at risk ofdeveloping cancer and methods of treating cancer in a subject in need ofsuch treatment. In one embodiment, the cancer is selected from the groupconsisting of prostate cancer, multiple myeloma, gliobastoma multiforme,and melanoma. In one embodiment, the cancer is melanoma.

In one embodiment, a vaccine composition of the invention isadministered to a subject having a cancer associated with overexpressionof MICA. The overexpression of MICA can be determined using any knownmethod in the art for measuring the expression level of a protein or thecorresponding nucleic acid. Such methods include, but are not limitedto; western blots, northern blots, southern blots, ELISA,immunoprecipitation, immunofluorescence, flow cytometry,immunocytochemistry, nucleic acid hybridization techniques, nucleic acidreverse transcription methods, and nucleic acid amplification methods.In one embodiment, the cancer is selected from the group consisting ofmelanoma, lung, breast, kidney, ovarian, prostate, pancreatic, gastric,and colon carcinoma, lymphoma or leukemia. In one embodiment, the canceris melanoma. In one embodiment, the cancer is a plasma cell malignancy,for example, multiple myeloma (MM) or pre-malignant condition of plasmacells. In some embodiments the subject has been diagnosed as having acancer or as being predisposed to cancer.

The vaccine compositions of the invention may be administered separatelyor as part of a therapeutic regimen or combination therapy, as describedbelow. The vaccine compositions of the invention may also beadministered singly, or in multiple administrations, for example in aprime-boost strategy. In this context, the term “prime-boost” refers tothe use of two different immunogens in succession. The two differentimmunogens are typically administered successively following a period oftime such as 10 to 30 days or 10 to 60 days. In one embodiment, theperiod of time is from 2 to 4 weeks. Thus, for example, in oneembodiment a vaccine composition of the invention is administered attime zero and a second vaccine composition of the invention (comprisinga different immunogen) is administered following a period of time, forexample from 10 to 30 days, from 10 to 60 days, or from 2 to 4 weeks.

The first and second vaccine compositions can be, but need not be, thesame composition. Thus, in one embodiment of the present invention, thestep of administering the vaccine comprises administering a firstvaccine composition, and then at a later time, administering a secondvaccine composition.

In one embodiment, one or a plurality of different vaccine compositionsof the invention is administered to the subject at multiple sites asdescribed in U.S. Pat. No. 8,110,196. Preferably, each site drains to alymph node or group of lymph nodes. In one embodiment, a vaccinecomposition of the invention is administered to multiple sites drainingto two or more lymph nodes selected from the group consisting of thelymph nodes of the head and neck, the axillary lymph nodes, thetracheobronchial lymph nodes, the parietal lymph nodes, the gastriclymph nodes, the ileocolic lymph nodes, and the inguinal and subinguinallymph nodes. In another embodiment, the sites are selected from thegroup consisting of the right arm, the left arm, the right thigh, theleft thigh, the right shoulder, the left shoulder, the right breast, theleft breast, the abdomen, the right buttock, and the left buttock. Inone embodiment, the site is or drains to a nonencapsulated cluster oflymphoid tissue selected from the group consisting of the tonsils, theadenoids, the appendix, and Peyer's patches. In one embodiment, avaccine composition of the invention is administered to a site thatdrains to the spleen.

In one embodiment, each vaccine composition is administered by a routeindependently selected from the group consisting of intradermally,subcutaneously, transdermally, intramuscularly, orally, rectally,vaginally, by inhalation, and a combination thereof. In one embodiment,at least one composition is injected directly into an anatomicallydistinct lymph node, lymph node cluster, or nonencapsulated cluster oflymphoid tissue.

Any suitable route of administration is encompassed by the methods ofthe invention, e.g. intradermal, subcutaneous, intravenous,intramuscular, or mucosal. Mucosal routes of administration include, butare not limited to, oral, rectal, vaginal, and nasal administration. Ina preferred embodiment, at least one composition is administeredtransdermally, intradermally, subcutaneously, orally, rectally,vaginally or by inhalation. Any route approved by the Food and DrugAdministration (FDA) can be used for the vaccine compositions of theinvention. Exemplary methods of administration are described in theFDA's CDER Data Standards Manual, version number 004 (which is availableat fda.give/cder/dsm/DRG/drg00301.htm).

Preferably, the route of administration is selected to target acomposition to a particular site, for example, by injection directlyinto a lymph node or a lymph node cluster, by oral administration totarget the lymph nodes of the stomach, by anal administration to targetthe lymph nodes of the rectum, by inhalation or aerosol to target thelymph nodes of the lungs, or by any other suitable route ofadministration.

Where the methods of the invention comprise administering a vaccinecomposition to multiple sites, each composition is preferablyadministered at substantially the same time, for example, within one toeight hours or during the same doctor's visit. In one embodiment, eachcomposition is administered within one to two hours, within one to threehours, within one to four hours, or within one to five hours.

Where the vaccine composition is in the form of a scaffold, the methodof vaccinating a subject comprises implanting the scaffold compositionin the subject, preferably subcutaneous implantation. In certainembodiments, the method of vaccinating a subject may comprise implantingor injecting the scaffold vaccine composition in two or more areas ofthe subject's anatomy.

In one embodiment, the methods of the invention further compriseadministering to the subject antigen presenting cells which have beensensitized with at least one MIC peptide. In a preferred embodiment, theantigen presenting cells are dendritic cells.

In one embodiment, the method further comprises administering to thesubject one or more adjuvants. In one embodiment, the one or moreadjuvants is selected from the group consisting of an oil-basedadjuvant, a CpG DNA adjuvant, polyinosinic:polycytidylic acid (usuallyabbreviated poly(I:C)), a mineral salt adjuvant, a mineral salt geladjuvant, a particulate adjuvant, a microparticulate adjuvant, a mucosaladjuvant, and a cytokine. Such adjuvants may either be formulated withthe compositions of the invention or administered separately from thecompositions, e.g., prior to, concurrently with, or after thecompositions are administered to the subject. The one or more adjuvantscan be covalently linked to the peptide or fusion protein of theinvention. For example, a CpG DNA adjuvant is covalently linked to thepeptide or fusion protein of the invention.

The methods disclosed herein can be applied to a wide range of species,e.g., humans, non-human primates (e.g., monkeys), horses, cattle, pigs,sheep, deer, elk, goats, dogs, cats, mustelids, rabbits, guinea pigs,hamsters, rats, and mice.

The terms “treat” or “treating,” as used herein, refers to partially orcompletely alleviating, inhibiting, ameliorating, and/or relieving thedisease or condition from which the subject is suffering. In someinstances, treatment can result in the continued absence of the diseaseor condition from which the subject is suffering.

In general, methods include selecting a subject at risk for or with acondition or disease. In some instances, the subject's condition ordisease can be treated with a pharmaceutical composition disclosedherein. For example, in some instances, methods include selecting asubject with cancer, e.g., wherein the subject's cancer can be treatedby targeting one or both of MICA.

In some instances, treatment methods can include a singleadministration, multiple administrations, and repeating administrationas required for the prophylaxis or treatment of the disease or conditionfrom which the subject is suffering. In some instances, treatmentmethods can include assessing a level of disease in the subject prior totreatment, during treatment, and/or after treatment. In some instances,treatment can continue until a decrease in the level of disease in thesubject is detected.

The terms “administer,” “administering,” or “administration,” as usedherein refers to implanting, absorbing, ingesting, injecting, orinhaling, the inventive peptide, regardless of form. In some instances,one or more of the peptides disclosed herein can be administered to asubject topically (e.g., nasally) and/or orally. For example, themethods herein include administration of an effective amount of compoundor compound composition to achieve the desired or stated effect.Specific dosage and treatment regimens for any particular patient willdepend upon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, and the judgment of the treating physician.

Following administration, the subject can be evaluated to detect,assess, or determine their level of disease. In some instances,treatment can continue until a change (e.g., reduction) in the level ofdisease in the subject is detected.

Upon improvement of a patient's condition (e.g., a change (e.g.,decrease) in the level of disease in the subject), a maintenance dose ofa compound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of disease symptoms.

In some instances, the disclosure provides methods for detecting immunecells e.g., B cells and/or memory B cells, from a human subject. Suchmethods can be used, for example, to monitor the levels of immune cellse.g., B cells and/or memory B cells, in a human subject, e.g., followingan event. Exemplary events can include, but are not limited to,detection of diseases, infection; administration of a therapeuticcomposition disclosed herein, administration of a therapeutic agent ortreatment regimen, administration of a vaccine, induction of an immuneresponse. Such methods can be used clinically and/or for research.

Effective Amounts and Dosages

In one embodiment, an effective amount of a vaccine composition of theinvention is the amount sufficient to reduce the severity of a cancer ina subject having cancer, or the amount sufficient to reduce orameliorate the severity of one or more symptoms thereof, the amountsufficient to prevent the progression of the cancer, the amountsufficient to prevent further metastasis of the cancer, the amountsufficient to cause clinical regression of the cancer, or the amountsufficient to enhance or improve the therapeutic effect(s) of anothertherapy or therapeutic agent administered concurrently with, before, orafter a vaccine composition of the invention.

Symptoms of cancer are well-known to those of skill in the art andinclude, without limitation, unusual mole features, a change in theappearance of a mole, including asymmetry, border, color and/ordiameter, a newly pigmented skin area, an abnormal mole, darkened areaunder nail, breast lumps, nipple changes, breast cysts, breast pain,death, weight loss, weakness, excessive fatigue, difficulty eating, lossof appetite, chronic cough, worsening breathlessness, coughing up blood,blood in the urine, blood in stool, nausea, vomiting, liver metastases,lung metastases, bone metastases, abdominal fullness, bloating, fluid inperitoneal cavity, vaginal bleeding, constipation, abdominal distension,perforation of colon, acute peritonitis (infection, fever, pain), pain,vomiting blood, heavy sweating, fever, high blood pressure, anemia,diarrhea, jaundice, dizziness, chills, muscle spasms, colon metastases,lung metastases, bladder metastases, liver metastases, bone metastases,kidney metastases, and pancreatic metastases, difficulty swallowing, andthe like.

In one embodiment, the effective amount of a vaccine composition of theinvention is the amount sufficient to produce an antibody secreting Bcell or cytotoxic T cell mediated immune response directed against oneor more of the peptides of the vaccine compositions of the invention. Inone embodiment, the effective amount of a vaccine composition of theinvention is the amount sufficient to produce an antibody secreting Bcell or cytotoxic T cell mediated immune response directed against acancer cell. The ability of the vaccine compositions of the invention toelicit an immune response can be determined using any routine methodavailable to those of skill in the art. In one embodiment, the effectiveamount of each composition is the amount sufficient to produce acytotoxic T cell response in the subject as measured, for example, by amixed lymphocyte T cell assay.

In one embodiment, the effective amount of the vaccine compositionadministered to the subject, or at a particular site of the subject, isthat amount which delivers 1 to 1000 micrograms of the one or morepeptides of the composition. In one embodiment, the amount of peptidesis 1 to 100 micrograms, 1 to 200 micrograms, 1 to 300 micrograms, 1 to400 micrograms, 1 to 500 micrograms, 1 to 600 micrograms, 1 to 700micrograms, 1 to 800 micrograms, or 1 to 900 micrograms. In anotherembodiment, the amount of peptides is 1 to 10 micrograms, 1 to 20micrograms, 1 to 30 micrograms, 1 to 40 micrograms, 1 to 50 micrograms,1 to 60 micrograms, 1 to 70 micrograms, 1 to 80 micrograms, or 1 to 90micrograms. In one embodiment, the total amount of peptides administeredto a subject does not exceed 5 milligrams. In one embodiment, the totalamount of peptides administered to a subject does not exceed 2milligrams.

Combination Therapy

The present invention also provides methods for the treatment orprophylaxis of cancer which comprise administering a vaccine compositionof the invention to a subject in need thereof, along with one or moreadditional therapeutic agents or therapeutic regimens. In oneembodiment, a vaccine composition of the invention is administered aspart of a therapeutic regimen that includes surgery, a chemotherapeuticagent, or radiation therapy, an immunotherapy, or any combination of theforegoing.

In one embodiment, the therapeutic regimen comprises or furthercomprises one or more immunostimulatory agents. In one embodiment, theone or more immunostimulatory agents is selected from the groupconsisting of an anti-CTLA-4 antibody or peptide, an anti-PD-1 antibodyor peptide, an anti-PDL-1 antibody or peptide, an anti-OX40 (also knownas CD134, TNFRSF4, ACT35 and/or TXGP1L) antibody or peptide, ananti-GITR (also known as TNFRSF18, AITR, and/or CD357) antibody orpeptide, an anti-LAG-3 antibody or peptide, and/or an anti-TIM-3antibody or peptide.

In one embodiment, the one or more immunostimulatory agents is selectedfrom an anti-MICA antibody described in WO 2013/049517 or WO2008/036981. In one embodiment, the one or more immunostimulatory agentsis selected from CM33322 Ab4, CM33322 Ab28, and CM33322 Ab29, which aredescribed in U.S. Provisional Application Nos. 61/792,034 and 61/913,198and in U.S. application Ser. No. 14/025,573.

In one embodiment, the therapeutic regimen comprises or furthercomprises one or more cytokines. In one embodiment, the vaccinecompositions of the invention comprise one or more cytokines. In oneembodiment, at least one cytokine is an interleukin or an interferon. Inone embodiment, at least one cytokine is an interleukin selected fromthe group consisting of IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, and IL-18. Inanother embodiment, at least one cytokine is an interferon selected fromIFN.alpha., IFN.beta., and IFN.gamma.

In one embodiment, a vaccine composition of the invention isadministered as part of a therapeutic regimen that includesadministering to the subject at least one chemotherapeutic agentselected from the group consisting of histone deacetylase inhibitors(“HDAC”) inhibitors, proteasome inhibitors, alkylating agents, andtopoisomerase inhibitors.

In one embodiment, the chemotherapeutic agent is an HDAC inhibitorselected from the group consisting of hydroxamic acid, Vorinostat(Zolinza), suberoylanilide hydroxamic acid (SAHA)(Merck), Trichostatin A(TSA), LAQ824 (Novartis), Panobinostat (LBH589) (Novartis), Belinostat(PXD101)(CuraGen), ITF2357 Italfarmaco SpA (Cinisello), Cyclictetrapeptide, Depsipeptide (romidepsin, FK228) (GloucesterPharmaceuticals), Benzamide, Entinostat (SNDX-275/MS-275)(SyndaxPharmaceuticals), MGCD0103 (Celgene), Short-chain aliphatic acids,Valproic acid, Phenyl butyrate, AN-9, pivanex (Titan Pharmaceutical),CHR-3996 (Chroma Therapeutics), and CHR-2845 (Chroma Therapeutics).

In one embodiment, the chemotherapeutic agent is a proteasome inhibitorselected from the group consisting of Bortezomib, (MillenniumPharmaceuticals), NPI-0052 (Nereus Pharmaceuticals), Carfilzomib(PR-171) (Onyx Pharmaceuticals), CEP 18770, and MLN9708.

In one embodiment, the chemotherapeutic agent is an alkylating agentsuch as mephalan.

In one embodiment, the chemotherapeutic agent is a topoisomeraseinhibitor such as Adriamycin (doxorubicin).

In one embodiment, the therapeutic regimen comprises or furthercomprises one or more of chemotherapy, radiation therapy, cytokines,chemokines and other biologic signaling molecules, tumor specificvaccines, cellular cancer vaccines (e.g., GM-CSF transduced cancercells), tumor specific monoclonal antibodies, autologous and allogeneicstem cell rescue (e.g., to augment graft versus tumor effects), othertherapeutic antibodies, molecular targeted therapies, anti-angiogenictherapy, infectious agents with therapeutic intent (such as tumorlocalizing bacteria) and gene therapy.

Kits

The invention provides a pharmaceutical pack or kit for carrying out themethods or therapeutic regimens of the invention. In one embodiment, thekit comprises a vaccine composition of the invention in lyophilizedform. In one embodiment, the kit comprises a vaccine composition of theinvention in the form of a protein scaffold.

In another embodiment, the kit further comprises in one or moreadditional containers a cytokine or an adjuvant.

The composition in each container may be in the form of apharmaceutically acceptable solution, e.g., in combination with sterilesaline, dextrose solution, or buffered solution, or otherpharmaceutically acceptable sterile fluid. Alternatively, thecomposition may be lyophilized or desiccated; in this instance, the kitoptionally further comprises in a separate container a pharmaceuticallyacceptable solution (e.g., saline, dextrose solution, etc.), preferablysterile, to reconstitute the composition to form a solution forinjection purposes.

In another embodiment, the kit further comprises one or more reusable ordisposable device(s) for administration (e.g., syringes, needles,dispensing pens), preferably packaged in sterile form, and/or a packagedalcohol pad. Instructions are optionally included for administration ofthe compositions by a clinician or by the patient. The kit may alsocomprise other materials, e.g., metal or plastic foil, such as a blisterpack.

In some embodiments, the present disclosure provides methods for usingany one or more of the vaccine compositions (indicated below as ‘X’)disclosed herein in the following methods.

Substance X for use as a medicament in the treatment of one or morediseases or conditions disclosed herein (e.g., cancer, referred to inthe following examples as ‘Y’). Use of substance X for the manufactureof a medicament for the treatment of Y; and substance X for use in thetreatment of Y.

In some instances, therapeutic compositions disclosed herein can beformulated for sale in the US, import into the US, and/or export fromthe US.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

EXAMPLES Example 1: General Methods

Vector Construction

Multivalent vaccines induce substantially higher-titer antibodyresponses than monovalent proteins. Herein is used a multivalent displayin which the MICA alpha3 domain is fused to Helicobacter pylori (H.pylori) ferritin. Ferritin-based nanoparticles were recently shown toinduce high-titer antibodies for influenza and EBV vaccines. Ferritin isfound in most organisms as an iron storage protein. Ferritin is aself-assembling particle that forms a spherical particle with octahedralsymmetry, consisting of 24 subunits. See FIGS. 1C and 1D for a schematicof a ferritin particle as well as a map demonstrating cellular andhumoral immune responses against human and mouse ferritin. MICA alpha3ferritin fusion gene (abbreviated as MICA-ferritin) was generated byfusing the gene encoding for α3 domain of MICA to H. pylori ferritinusing a Gly-Ser-Gly linker (FIG. 2A). A point mutation (Asn19Gln) wasintroduced in the H. pylori ferritin to abolish a potentialN-glycosylation site. To determine the antibody response of MICAα3 alone(without the ferritin), deglycosylated version of MICAα3 gene wasgenerated by mutating 7 out of 8 potential N-glycosylation sites to Aspor Gln. A C-terminal HA tag was included for downstream proteinpurification purpose. The genes were synthesized using GeneArt® GeneSynthesis platform and codon optimized for insect cell expression. Thesynthesized genes were cloned into pAcDB3 baculovirus expression vector.(See FIG. 2C).

Assays that demonstrate the generation of MIC alpha 3 domain vaccine andthe generation of deglycosylated MICA alpha3 vaccine are presented inFIGS. 2B and 2D, respectively.

Protein Biosynthesis and Purification

MICAα3 and MICA-ferritin fusion proteins were expressed in Sf9(Spodoptera frugiperda) insect cells by infecting these cells withrecombinant baculovirus at a multiplicity of infection of 10. The cellswere grown in Sf900 serum free expression medium (Life Technologies) andthe cultured supernatants were collected 3 days post-transfection. Thesupernatants were concentrated and then exchanged into Tris buffer (50mM Tris, 150 mM NaCl, pH 7.5 buffer). The proteins were purified by HAaffinity chromatography and aggregates were removed by performing sizeexclusion chromatography using Superose 6 column (GE Healthcare). Thepurified proteins were buffer exchanged into PBS using PD-10 desaltingcolumns (GE) and concentrated to 1 mg/ml using Amicon Ultra 4 mlcentrifugal filters. Protein purity and size was verified by SDS-PAGE.

Preparation of MPS Vaccine and Immunization

The scaffold vaccine described here was recently reported (Kim et alNat. Biotechnol. 2015, 33, 64-72). Mesoporous silica rods (MSR) injectedwith a needle spontaneously assembles in vivo to form a macroporousstructure resembling a haystack that provides a 3D cellularmicroenvironment for dendritic cells. This biodegradable scaffoldrecruits and educates dendritic cells which then migrate to lymph nodeswhere they induce an immune response. 5 mg of MSR was loaded with 1 μgof GM-CSF (to recruit dendritic cells), 100 μg of CpG oligonucleotide(to induce dendritic cell activation) and 200 μg of MICA-ferritin fusionor a control protein (ovalbumin) for 12 hours at room temperature. Theparticles were lyophilized, resuspended in PBS and injectedsubcutaneously into the flank of C57BL/6 mice. Mice receive a boost ondays 14 or 21 following initial immunization. Age matched non-immunizedmice (naïve) and mice immunized with ovalbumin were be used as controlgroups for the MICA-ferritin immunization experiments. As an additionalcontrol in the MICAα3 experiment, mice were immunized with all vaccinecomponents but without the MSR scaffold (bolus).

Lung Metastasis Experiment in MICAα3 and MICA Ferritin Immunized MiceC57Bl/6J mice were immunized with MICAα3 or MICAα-ferritin vaccine.Three weeks after the boost, mice were challenged with intra venous(i.v) injection of 0.5×10⁶ MICA expressing B16F10 melanoma cells. Serawere collected prior to tumor challenge and at weekly intervals toanalyze shed MICA levels. Mice were euthanized 14 days after tumorchallenge; lungs were harvested and fixed in 10% neutral-bufferedformalin and the number of pulmonary metastases was quantified.

Flow Cytometric Analysis and ELISA to Determine the MICA Antibody Titersin Immunized Mice

MICA specific antibody titers were tested by ELISA using the full lengthextracellular domain of MICA. Full length MICA protein (0.2m) was coatedon 96 well ELISA plate overnight at 4° C. The plates were blocked for anhour at room temperature with PBS/2% BSA. The plates were washed andincubated with serial dilution of sera collected at weekly intervalsfrom each experimental group. Goat anti-mouse HRP was used as detectionantibody. Flow cytometric analysis was used to assess the binding ofserum antibodies to full length expressed on the surface of tumor cells.Briefly, 1×10⁵ tumor cells were incubated with 1 μl of serum for 2 hrsat 4° C. 1×10⁵ cells were stained with 1 μl of serum from non-immunizedmice (naïve), mice immunized with vaccine components without the MSRscaffold (bolus) or MICA-ferritin vaccine (vaccinated) in 100μ1 of PBSfor 2 hours. Commercially available monoclonal antibody 6D4 that bindsto the alpha1-alpha2 domains of MICA was used as a positive control(10m). PE conjugated anti-mouse IgG was used as secondary antibody.

Example 2: Scaffold Vaccine for Induction of Potent Immune Response

The MIC α3 domain was expressed as a recombinant protein in theBaculovirus system; the protein was displayed in a multivalent form onH. pylori ferritin, an iron storage protein with 24 identical subunits.A vaccination approached using mesoporous silica rods (MSRs) originallydescribed in Kim et al Nat. Biotechnol. 2015, 33, 64-72 was used herein,and is hereby incorporated by reference in its entirety (see FIG. 3).MSRs that are injected subcutaneously with a needle spontaneouslyassemble in vivo into macroporous structures that provide a 3D cellularmicroenvironment for host immune cells. This system recruits largenumbers of immune cells, exposes them to the relevant antigens and alsoprovides the appropriate molecular cues for induction of a potent immuneresponse. The MIC α3 domain protein was absorbed to MSRs, along withGM-CSF (for recruitment of dendritic cells) and CpG oligonucleotide (anadjuvant that activates dendritic cells). This vaccination approachenabled induction of high-titer antibodies specific to the MIC α3domain. These antibodies stained tumor cells that express MIC andinhibited shedding of MIC by tumor cells.

To test the anti-tumor activity of this vaccine, we utilized B16melanoma cells transfected with human MIC. When these tumor cells areinjected intravenously into non-immunized mice, they form large numbersof lung metastases (˜200 metastases/mouse). The MSR scaffold vaccineprovided potent protection from the outgrowth of such metastases. Whenthe vaccine components were injected as a bolus without the MSRscaffold, partial protection was observed, but the biological effect wassignificantly weaker. This result shows that local recruitment of immunecells to the MSR scaffold greatly enhances the activity of this vaccine.(See FIG. 4).

Example 3: Vaccination with MICA-Ferritin Fusion Protein InducesHigh-Titers of MICA Specific Antibodies

Binding of MICAα3 specific antibodies in the sera of immunized mice tofull length MICA expressed on the surface of B16F10 mouse melanoma cellswas tested by flow cytometry. Briefly, 1×10⁵ cells were stained with 1μl of serum from non-immunized mice (naïve), mice immunized with controlvaccine (OVA-protein) or MICA-ferritin vaccine from days 14, 28 and 42(vaccinated) in 100μ1 of PBS for 2 hours. Commercially availablemonoclonal antibody 6D4 that binds to the α1-α2 domains of MICA was usedas a positive control (10m). PE conjugated anti-mouse IgG was used assecondary antibody. MICAα3 specific antibodies in the sera of vaccinatedmice (histograms—green (d14), blue (d28), red (d42) showed significantbinding to MICA expressed on the tumor cell surface (FIGS. 5A and 5B).The results of these assays demonstrate that MICA-feritin fusion proteinvaccination induces high-titers of MICA specific antibodies.

Example 4: Vaccination with MICA-Ferritin Fusion Protein Generates HighLevels of IGG1, Igg2a and IGG3 Micaa3 Specific Polyclonal AntibodyResponse

Sera from MICA-ferritin immunized mice were tested in ELISA to determinethe different subclasses of IgGs induced upon vaccination. Sera frommice immunized with OVA-protein (bolus) or non-immunized mice (naive)were used as control groups. Full length MICA was used as the captureantigen and a serum dilution of 1/1000 was used in each well. HRPconjugated anti-mouse IgG1, IgG2a, IgG2b or IgG3 were used fordetection. Immunization with MICA-ferritin (vaccinated) was found toinduce high levels of all the IgG subclasses tested (See FIG. 6).

Example 5: Polyclonal Antibodies Generated in Response to theMICA-Ferritin Vaccine Prevent MICA Shedding from the Surface of HumanMetastatic Melanoma Cell Line

Induction of MICA antibodies in melanoma patients treated withautologous tumor vaccine (GVAX) plus Ipilimumab, correlated with reducedserum soluble MICA (sMICA) levels. The extracellular part of MICAScontains two MEW class I-like domains (α1 and α2) and amembrane-proximal immunoglobulin domain (α3). It has been shown that thedisulfide isomerase ERp5 cleaves the structural disulfide bond in theMICA α3 domain, and the resulting unfolding of this domain allowsproteolytic cleavage by ADAM 10, ADAM 17 and MMP-14. The purpose of thisassay was to determine whether the polyclonal antibodies generated inresponse to the MICA-ferritin vaccine prevents MICA shedding from thehuman melanoma tumor cell line A375.

For these assays, 4×10⁵ A375 malignant melanoma cells were plated in 96well plate with 200μ1 of media. The cells were incubated with no serumor with serum from naïve, OVA-protein immunized or MICA-ferritinvaccinated mice (FIG. 7 bars with inverted triangle) for 24 hrs. sMICAin the supernatant was analyzed using MICA ELISA kit which utilizes MICAα1-α2 domain antibodies for capture and detection. Lower levels of sMICAwas detected in the supernatant of cells incubated with serum fromMICA-ferritin vaccinated mice (FIG. 7 bars with inverted triangle)compared to cells that were incubated without serum, serum from naïve(FIG. 7 bars with circles and squares) or OVA-protein immunized mice(FIG. 7 bars with triangle), thus indicating that the MICAα3 specificantibodies can inhibit the shedding of MICA from tumor cell surface.

Example 6: Therapeutic Activity of MICA-Ferritin Vaccine

The therapeutic activity of the MICA-ferritin vaccine was tested usinghighly aggressive B16F10 melanoma tumor model. B16F10 melanoma tumorcells were genetically modified to express human MICA. MICA is bound bythe murine NKG2D receptor, making this a suitable model system. C57BL/6mice immunized with the MICA-ferritin vaccine, OVA-protein vaccine(control antigen) and non-immunized control mice (age and sex matched)were challenged with i.v injection of B16F10-MICA tumor cells. Sera werecollected prior to tumor challenge, on day 7 and day 13. Mice wereeuthanized 14 days after tumor challenge and the number of pulmonarymetastases was quantified (See FIG. 8A).

For these assays, 8 week old C57BL/6 female mice were immunized withMICA alpha3-ferritin or ova-protein followed by a boost on day 28. Threeweeks later, mice were challenged by i.v. of 5×10⁵ MICA-expressingB16F10 melanoma cells. Mice were euthanized 14 days after tumorchallenge and the number of pulmonary metastases was quantified. ShedMICA (sMICA) level in the sera was monitored by ELISA. These experimentsdemonstrated that MICA-ferritin vaccinated mice were nearly tumor free.In contrast, non-immunized age-matched control group (naïve) and micevaccinated with control antigen—ovalbumin had large numbers of lungmetastases (average of ˜150 lung mets/mouse) (FIG. 8A). Importantly,sMICA was undetectable in sera of mice immunized with MICA-ferritinvaccine (triangle) while high levels of sMICA were detected within twoweeks after tumor challenge in the sera of mice immunized with ovalbumin(square) and the non-immunized control group (circle) (FIG. 8B).

Example 7: Determining Effective Dosage of MICA-Ferritin Vaccine andKinetics of Polyclonal Antibody Response In Vivo

For these studies, mice received two injections of the vaccine prior totumor cell challenge. However, near-maximal antibody levels are alreadyachieved two weeks following initial immunization. In order to determinethe optimal vaccination dosage and kinetics of polyclonal antibodyresponse at different doses, C57Bl/6J mice were immunized differentdoses of MICA-ferritin protein (50-200m) absorbed to MSR. The micereceived boost on day 17. Sera were collected at weekly intervals byretro-orbital bleeding to determine the MICA antibody titers by ELISA.On day 25 following initial immunization, mice were challenged with i.v.injection of MICA expressing B16F10 melanoma cells. Sera were collectedprior to tumor challenge and at weekly intervals to analyze shed MICAlevels. Mice were euthanized 14 days after tumor challenge; lungs wereharvested and fixed in 10% neutral-buffered formalin and the number ofpulmonary metastases was quantified.

For these studies, 8 week old C57BL/6 female mice were immunized withMICA alpha3-ferritin vaccine at different doses (50 μg, 100 μg or 200μg) and boosted on day 17. End point antibody titer was determined byserially diluting the sera and testing its binding to full length MICAprotein by ELISA. MICA-ferritin immunized mice elicited high levels ofantibody titers by day 14 (ELISA endpoint titers of 10⁵) at all dosestested and the titer increased by ˜1000 fold after boost on day 17.Naïve, untreated age matched mice were used as control group (See FIG.9A).

On day 25 following initial immunization, mice were challenged with i.v.injection of 0.5×10⁶ MICA expressing B16F10 melanoma cells. Sera werecollected prior to tumor challenge and at weekly intervals to analyzeshed MICA levels. Mice were euthanized 14 days after tumor challenge;lungs were harvested and fixed in 10% neutral-buffered formalin and thenumber of pulmonary metastases was quantified. Mice immunized with 100μg and 200 μg were nearly tumor free compared to mice immunized with 50μg of the vaccine (˜2-12 lung mets). sMICA was undetectable in the seraof mice immunized with different doses of MICA-ferritin vaccine (50μg—square, 100 μg—upward triangle, 200 μg—downward triangle) while highlevels of sMICA were detected within two weeks after tumor challenge inthe sera non-immunized control group (empty circle) (see FIGS. 9B and9C).

Example 8: MICAA3 Vaccine Alone Induces High-Titers of Mica SpecificAntibodies

To determine the effect of MICAα3 vaccine alone (without ferritin) ingenerating MICA specific polyclonal antibody response, deglycosylatedversion of MICAα3 gene was generated by mutating 7 out of 8 potentialN-glycosylation sites to Asp or Gln. Following protein production andpurification as described in the methods section, MICAα3 vaccine wasprepared by loading 5 mg of MSR with 1 μg GM-CSF, 100 μg CpG-ODN and 150μg of deglycosylated MICAα3 protein (abbreviated MICAα3 vaccine). Theparticles were then lyophilized, re-suspended in cold PBS (150 μl) andinjected subcutaneously into the flank of female C57Bl/6J mice. Miceimmunized with all vaccine components but without the MSR scaffold(bolus) and untreated, age matched mice were used as control groups.Sera were collected at weekly intervals by retro-orbital bleeding. Themice received boost on day 28 following initial immunization.

For these studies, 1×10⁵ MICA009 expressing B16F10 melanoma cells werestained with 1 μl of serum from non-immunized mice (naïve), miceimmunized with MICAα3 without MSR (bolus) or MICAα3 vaccine (vaccinated)in 100μ1 of PBS for 2 hours. Commercially available monoclonal antibody6D4 that binds to the α1-α2 domains of MICA was used as a positivecontrol (10 μg). PE conjugated anti-mouse IgG was used as secondaryantibody. MICAα3 specific antibodies in the sera of vaccinated mice andbolus group showed significant binding to MICA expressed on the tumorcell surface, with levels similar to the positive control groupfollowing boost (See FIG. 10A).

Sera from MICAα3 immunized mice were tested in ELISA to determine thedifferent subclasses of IgGs induced upon vaccination. Sera fromnon-immunized mice were used as control groups. Full length MICA wasused as the capture antigen and a serum dilution of 1/1000 was used ineach well. HRP conjugated anti-mouse IgG1, IgG2a, IgG2b or IgG3 wereused for detection. Immunization with MICAα3 vaccine and the bolusvaccine were found to induce the production of all the IgG subclassestested, with IgG1 levels higher than the MICA-ferritin vaccine (See FIG.10B).

Example 8: MICAA3 Vaccine Alone (without Ferritin Fusion) ShowsSignificant Therapeutic Benefit In Vivo

For these studies, 8 week old C57Bl/6J female mice were immunized withMICAα3 vaccine or bolus consisting of all the vaccine components butwithout the MSR scaffold. Untreated, age matched C57Bl/6J female micewere used as the control group. Three weeks after the boost, mice werechallenged with i.v. injection of 0.5×10⁶ MICA expressing B16F10melanoma cells. Mice were euthanized 14 days after tumor challenge;lungs were harvested and fixed in 10% neutral-buffered formalin and thenumber of pulmonary metastases was quantified. MICAα3 vaccinated micewere nearly tumor free compared to untreated, age matched control group.The number of pulmonary metastases was significantly lower in the bolusgroup (˜100-125) compared to the non-immunized group (˜200-250) (SeeFIG. 11A).

sMICA was undetectable in sera of mice immunized with MICAα3 vaccine(triangle) while elevated levels of sMICA was found in the untreatedcontrol group within two weeks after tumor challenge (circle). The bolusgroup had relatively lower levels of sMICA in the sera (square) comparedto the control group. (See FIG. 11B). The increased number of lungmetastases and sMICA levels in mice immunized with MICAα3 bolus comparedto the vaccinated group is most likely due to observed levels ofreduction in MICA specific antibody titers by day 62 following initialimmunization compared to the vaccinated group (data not shown).

Example 9: Determining Cytotoxic Lymphocyte Populations that areRequired for Vaccine Efficacy

By depleting CD8 T cells or NK cells with mAbs, it was found that bothCD8 T cells and NK cells contribute to the therapeutic effect of thevaccine (FIGS. 13A-13B and 14A-14B).

Example 10: ELISA Assay for Quantification of MIC Antibodies

An ELISA assay for quantification of MIC antibodies induced by thevaccine is used (FIG. 6).

Example 11: Future Studies

The following are future studies to be performed to further assessvaccine performance.

1. Vaccine formulations will be optimized by testing the optimal amountof antigen and comparing two adjuvants, CpG oligonucleotide andPoly(I:C).

2. The efficacy of the vaccine will be tested in multiple tumor models,specifically the B16-MIC melanoma model (both subcutaneous andmetastasis models) and the orthotopic TRAMP-MIC model of prostatecancer. These studies involve measurement of vaccine efficacy byassessing the inhibition of tumor growth and the reduction of shed MICin the serum.

3. Sera will be transferred from immunized mice to non-immunizedrecipients to examine if the induced MIC-specific antibodies aresufficient for the protection afforded by the vaccine.

4. Determination of whether the vaccine provides protection againstsecondary challenge by tumor cells that lack MIC expression due toinduction of a CD8 T cell response against other tumor antigens. Micethat survive the B16-MIC metastasis model will be challenged by i.v.injection of a high dose of B16 tumor cells that express or do notexpress MIC.

Further investigations of the biomarkers that reflect the mechanisticactivity of induced antibodies will be performed. These will include thefollowing approaches.

1. ELISA assay for shed MIC in serum; an assay is available and will berigorously tested with serum samples from patients with advanced cancer.

2. Testing of functional activity of induced MIC α3 domain antibodies.Which human tumor cell lines are optimal for assays that assessantibody-mediated inhibition of MIC shedding (a panel of cell lines isavailable) will be studied.

3. Flow cytometry analysis of immune cells in peripheral blood and tumorbiopsies. Particularly important is the quantification of surface NKG2Dlevels by CD8 T cells and NK cells; antibodies are available and thepanel will be optimized.

Example 12: Baculovirus Expression of MICA002 Alpha 3 Fused to Ferritin(H. Pylori)

Purpose: Insect cell expression of MICA (002) alpha 3 fused to ferritin nanoparticle  General design: Signal peptide, 6 his tag (SEQ ID NO: 31), linker, N-terminal HA peptide,MICA alpha 3 domain (*002:01), GSG linker, H. pylori ferritin, stop codon   1 MVPCTLLLLL AAALAPTQTR AHHHHHHSKS YPYDVPDYAR signal peptide,     6HIS, linker, HA  41 TVPPMVNVTR SEASEGNITV TCRASGFYPW NITLSWRQDG MICA alpha 3  81 VSLSHDTQQW GDVLPDGNGT YQTWVATRIS QGEEQRFTCY 121 MEHSGNHSTH PVPSGKVLVL QSHWQTFHGS GDIIKLLNEQ linker, ferritin 161 VNKEMQSSNL YMSMSSWCYT HSLDGAGLFL FDHAAEEYEH 201 AKKLIIFLNE NNVPVQLTSI SAPEHKFEGL TQIFQKAYEH 241 EQHISESINN IVDHAIKSKD HATFNFLQWY VAEQHEEEVL 281 FKDILDKIEL IGNENHGLYL ADQYVKGIAK SRKS* (SEQ ID NO: 5) Strategy: Clone into pacDB3 vector between SmaI and BamHI sites Strategy: Clone into pacDB3 vector  Translation of DNAMAN18(1-945) Universal code  Total amino acid number: 314, MW = 35668Max ORF: 1-942, 314 AA, MW = 35668  1 ATGGTCCCCTGTACCCTGCTGCTGCTGCTGGCTGCTGCACTGGCACCTACTCAGACTCGG   1  M  V  P  C  T  L  L  L  L  L  A  A  A  L  A  P  T  Q  T  R  61 GCCCACCATCATCACCATCACTCAAAAAGTTACCCCTACGATGTCCCCGACTACGCCAGG  21  A  H  H  H  H  H  H  S  K  S  Y  P  Y  D  V  P  D  Y  A  R 121 ACCGTGCCCCCTATGGTGAACGTCACACGCTCAGAAGCTAGCGAGGGCAATATCACCGTG  41  T  V  P  P  M  V  N  V  T  R  S  E  A  S  E  G  N  I  T  V 181 ACATGCCGAGCATCTGGGTTCTATCCTTGGAACATTACACTGAGTTGGAGGCAGGACGGG  61  T  C  R  A  S  G  F  Y  P  W  N  I  T  L  S  W  R  Q  D  G 241 GTGTCCCTGTCTCACGATACTCAGCAGTGGGGCGACGTGCTGCCAGATGGCAATGGGACC  81  V  S  L  S  H  D  T  Q  Q  W  G  D  V  L  P  D  G  N  G  T 301 TACCAGACATGGGTGGCTACTCGGATCTCCCAGGGGGAGGAACAGAGATTCACCTGCTAT 101  Y  Q  T  W  V  A  T  R  I  S  Q  G  E  E  Q  R  F  T  C  Y 361 ATGGAGCATAGTGGAAACCACTCAACACATCCTGTGCCATCTGGCAAGGTGCTGGTCCTG 121  M  E  H  S  G  N  H  S  T  H  P  V  P  S  G  K  V  L  V  L 421 CAGAGTCACTGGCAGACATTTCATGGATCAGGCGATATCATTAAGCTGCTGAACGAACAG 141  Q  S  H  W  Q  T  F  H  G  S  G  D  I  I  K  L  L  N  E  Q 481 GTGAACAAGGAGATGCAGTCTAGTAACCTGTACATGAGCATGTCAAGCTGGTGTTATACA 161  V  N  K  E  M  Q  S  S  N  L  Y  M  S  M  S  S  W  C  Y  T 541 CACTCCCTGGACGGAGCCGGCCTGTTCCTGTTTGATCACGCCGCTGAGGAATACGAACAT 181  H  S  L  D  G  A  G  L  F  L  F  D  H  A  A  E  E  Y  E  H 601 GCTAAGAAACTGATCATTTTCCTGAATGAGAACAATGTGCCAGTCCAGCTGACTAGCATT 201  A  K  K  L  I  I  F  L  N  E  N  N  V  P  V  Q  L  T  S  I 661 TCCGCACCCGAACACAAGTTCGAGGGCCTGACCCAGATCTTTCAGAAAGCCTACGAACAC 221  S  A  P  E  H  K  F  E  G  L  T  Q  I  F  Q  K  A  Y  E  H 721 GAGCAGCATATCTCTGAAAGTATCAACAACATCGTGGACCACGCAATCAAGAGCAAAGAT 241  E  Q  H  I  S  E  S  I  N  N  I  V  D  H  A  I  K  S  K  D 781 CATGCCACCTTCAACTTTCTGCAGTGGTACGTGGCCGAGCAGCACGAGGAAGAGGTCCTG 261  H  A  T  F  N  F  L  Q  W  Y  V  A  E  Q  H  E  E  E  V  L 841 TTTAAGGACATTCTGGATAAAATCGAACTGATTGGCAATGAGAATCACGGGCTGTACCTG 281  F  K  D  I  L  D  K  I  E  L  I  G  N  E  N  H  G  L  Y  L 901 GCAGATCAGTATGTCAAGGGCATCGCAAAGTCAAGGAAATCATGA (SEQ ID NO: 6) 301  A  D  Q  Y  V  K  G  I  A  K  S  R  K  S  *  (SEQ ID NO: 7) SEQ DNAMAN: 945 bp;  Composition 254 A; 252 C; 241 G; 198 T; 0 OTHER Percentage: 26.9% A; 26.7% C; 25.5% G; 21.0% T; 0.0% OTHER Molecular Weight (kDa): ssDNA: 291.81 dsDNA: 582.6  ORIGIN   1 ATGGTCCCCT GTACCCTGCT GCTGCTGCTG GCTGCTGCAC TGGCACCTAC TCAGACTCGG  61 GCCCACCATC ATCACCATCA CTCAAAAAGT TACCCCTACG ATGTCCCCGA CTACGCCAGG 121 ACCGTGCCCC CTATGGTGAA CGTCACACGC TCAGAAGCTA GCGAGGGCAA TATCACCGTG 181 ACATGCCGAG CATCTGGGTT CTATCCTTGG AACATTACAC TGAGTTGGAG GCAGGACGGG 241 GTGTCCCTGT CTCACGATAC TCAGCAGTGG GGCGACGTGC TGCCAGATGG CAATGGGACC 301 TACCAGACAT GGGTGGCTAC TCGGATCTCC CAGGGGGAGG AACAGAGATT CACCTGCTAT 361 ATGGAGCATA GTGGAAACCA CTCAACACAT CCTGTGCCAT CTGGCAAGGT GCTGGTCCTG 421 CAGAGTCACT GGCAGACATT TCATGGATCA GGCGATATCA TTAAGCTGCT GAACGAACAG 481 GTGAACAAGG AGATGCAGTC TAGTAACCTG TACATGAGCA TGTCAAGCTG GTGTTATACA 541 CACTCCCTGG ACGGAGCCGG CCTGTTCCTG TTTGATCACG CCGCTGAGGA ATACGAACAT 601 GCTAAGAAAC TGATCATTTT CCTGAATGAG AACAATGTGC CAGTCCAGCT GACTAGCATT 661 TCCGCACCCG AACACAAGTT CGAGGGCCTG ACCCAGATCT TTCAGAAAGC CTACGAACAC 721 GAGCAGCATA TCTCTGAAAG TATCAACAAC ATCGTGGACC ACGCAATCAA GAGCAAAGAT 781 CATGCCACCT TCAACTTTCT GCAGTGGTAC GTGGCCGAGC AGCACGAGGA AGAGGTCCTG 841 TTTAAGGACA TTCTGGATAA AATCGAACTG ATTGGCAATG AGAATCACGG GCTGTACCTG 901 GCAGATCAGT ATGTCAAGGG CATCGCAAAG TCAAGGAAAT CATGA (SEQ ID NO: 8)Step 1. Amplify template for PCR 1 (signal peptide, 6 his (SEQ ID NO: 31),linker, HA, MICA a1pha3) from C1347 construct using primers Forward primer# ferritin_baculo_SmaIfor  (SEQ ID NO: 9)5′ AAAAAACCCGGGATGGTCCCCTGTACCCTGCTGCTGCTGC 3′ Internal reverse primer: # ferritin baculo_IRev  (SEQ ID NO: 10)5′ GTTCGTTCAGCAGCTTAATGATATCGCCTGATCCATGAAATGTCTGCCAG 3′ Step 2. Amplify template for PCR 2 (ferritin) from C1347 using Internal forward primer: # ferritin baculo_IF  (SEQ ID NO: 11)5′ CTGGCAGACATTTCATGGATCAGGCGATATCATTAAGCTGCTGAACGAAC 3′ Reverse primer: # ferritin baculo_BamHIRev  (SEQ ID NO: 12)5′ AAAAAAGGATCCTCATGATTTCCTTGACTTTGCGATGCCCTTG 3′ Step 3: Fusion PCR using primers  # ferritin_baculo_SmaIfor  and # ferritin_baculo_BamHIRev  Restriction analysis on DNAMAN18 Methylation: dam-No dcm-No  Screened with 117 enzymes, 18 sites found ApaI  1 GGGCC/C       63  BolI  2 T/GATCA       573   611 BglII  1 A/GATCT       695  BsiI  2 C/TCGTG       718   823 Bsp1407I 1 T/GTACA       509  BspHI  1 T/CATGA       940 (SEQ ID NO: 13) BspMI  1 ACCTGCNNNN/        361  (SEQ ID NO: 14)Eam1105I 1 GACNNN/NNGTC        240  Eco56I  1 G/CCGGC       556 (SEQ ID NO: 15) EcoNI  1 CCTNN/NNNAGG        841  EcoRV  1 GAT/ATC      456  NaeI  1 GCC/GGC       558  NheI  1 G/CTAGC       157 PstI   2 CTGCA/G       422   803  PvuII  1 CAG/CTG       648 List by Site Order   63 ApaI     456 EcoRV    611 BolI  803  PstI 157 NheI     509 Bsp1407I 648 PvuII 823  BsiI 240 Eam1105I 556 Eco56I   695 BglII 841  EcoNI 361 BspMI    558 NaeI     718 BsiI  940  BspHI  422 PstI     573 BolI Non Cut Enzymes  AatII   Acc65I   AccIII   AclI     AflII  AgeI AhaIII  Alw44I   AlwNI    ApaBI    ApaLI  AscI Asp718I AsuII    AvrII    BalI     BamHI  BbeI BbvII   BglI     Bpu1102I Bsc91I   BsmI   BspMII BssHII  BstD102I BstEII   BstXI    Bsu36I ClaI Csp45I  CspI     CvnI     DraI     DraIII DrdI EagI    Ecl136II Eco31I   Eco47III Eco52I Eco57I Eco72I  EcoICRI  EcoRI    EheI     EspI   FseI HindIII HpaI     I-PpoI   KpnI     MfeI   Mlu113I MluI    MscI     MstI     MstII    NarI   NcoI NdeI    NotI     NruI     NsiI     PacI   Pf1MI PinAI   PmaCI    PmeI     PvuI     RleAI  SadI SacII   SalI     SapI     Saul     ScaI   SoiI SfiI    SgrAI    SmaI     SnaBI    SpeI   SphI SplI    SpoI     SrfI     SspI     SstI   SstII StuI    SunI     SwaI     Tth111I  VspI   XbaI XcmI    XhoI     XmaI     XmaIII   XmnI   XorII Ferritin from H. Pylori  (SEQ ID NO: 16) MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWC YTHSLDGAGLFLFDHAAEEYEHAKKLIIF LNENNVPVQLTSISAPEHKFEGLIQIFQKAYEHEQHISESINNIVDHAIKSKDHATENFL QWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 181 Position N19 changed to Q (to eliminate N-linked glycosylation site), start at position 5 (underlined)  ferritin [Helicobacter pylori]NCBI Reference Sequence: WP_000949190.1  FASTA Graphics  Go to: LOCUS WP_000949190 167 aa linear BCT  May 16, 2013 DEFINITION ferritin [Helicobacter pylori].  ACCESSION WP_000949190 VERSION WP_000949190.1 GI: 446871934  KEYWORDS RefSeq. SOURCE Helicobacter pylori  ORGANISM Helicobacter pylori Bacteria; Proteobacteria; Epsilonproteobacteria;  Campylobacterales; Helicobacteraceae; Helicobacter. COMMENT REFSEQ: This record represents a single, non-redundant, protein  sequence which may be annotated on many different RefSeq genomes  from the same, or different, species. COMPLETENESS: full length.  FEATURES Location/Qualifiers source   1 . . . 167           /organism = “Helicobacter pylori”         /db_xref = “taxon:210” Protein  1..167          /product = “ferritin”          /calculated_mol_wt = 19183Region   3 . . . 158           /region_name = “Nonheme Ferritin”         /note = “nonheme-containing ferritins; cd01055”         /db_xref = “CDD:153113” Region   7 . . . 144          /region_name = “Ferritin”         /note = “Ferritin-like domain; pfam00210”         /db_xref = “CDD:249681”Site     order(17, 49 . . . 50, 53, 94, 126, 129 . . . 130)          /site_type = “other”         /note = “ferroxidase diiron center [ion binding]”         /db_xref = “CDD:153113” ORIGIN  (SEQ ID NO: 17)  1 mlskdiikll neqvnkemns snlymsmssw cythsldgag lflfdhaaee yehakkliif  61 lnennvpvql tsisapehkf egltqifqka yeheqhises innivdhaik skdhatfnfl 121 qwyvaeghee evlfkdildk ielignenhg lyladqyvkg iaksrks 

Example 11: Deglycosylated MICA 002 Protein Expression in Insect Cells

Purpose: Baculovirus expression of deglycosylated MICA alpha 3 (*002:01) General design: Signal peptide, N-terminal HA peptide, MICA alpha 3 domain (*002:01), stop codon(SEQ ID NO: 25)  1 MVPCTLLLLL AAALAPTQTR ASKSYPYDVP DYARTVPPMV QVTRSEASEG QITVTCRASG signal peptide, HA  61 FYPWNINLSW RQDGVSLSHD TQQWGDVLPD GNGTYQTWVA TRISQGEEQR FTCYMEHSGQ MICA alpha 3  121 HSTHPVPSGK VLVLQSHWQT FH* stop Strategy: Clone into pAcDB3 BglII-EcoRI site  SEQ DNAMAN1: 432 bp; Composition 96 A; 125 C; 122 G; 89 T; 0 OTHER Percentage: 22.2% A; 28.9% C; 28.2% G; 20.6% T; 0.0%0THER Molecular Weight (kDa): ssDNA: 133.37 dsDNA: 266.4  ORIGIN (SEQ ID NO: 18)  1 ATGGTCCCCT GTACCCTGCT GCTGCTGCTG GCTGCTGCAC TGGCACCTAC TCAGACTCGG  61 GCCTCAAAAA GTTACCCCTA CGATGTCCCC GACTACGCCA GGACCGTGCC CCCTATGGTG 121 CAGGTCACAC GCTCAGAAGC TAGCGAGGGC CAAATCACCG TGACATGCCG AGCATCTGGG 181 TTCTATCCTT GGAACATTAA CCTGAGTTGG AGGCAGGACG GGGTGTCCCT GTCTCACGAT 241 ACTCAGCAGT GGGGCGACGT GCTGCCAGAT GGCAATGGGA CCTACCAGAC ATGGGTGGCT 301 ACTCGGATCT CCCAGGGGGA GGAACAGAGA TTCACCTGCT ATATGGAGCA TAGTGGACAG 361 CACTCAACAC ATCCTGTGCC ATCTGGCAAG GTGCTGGTCC TGCAGAGTCA CTGGCAGACA 421 TTTCATTGA  Translation of DNAMAN1(1-432)  Universal code Total amino acid number: 143, MW = 15928Max ORF: 1-429, 143 AA, MW = 15928  1 ATGGTCCCCTGTACCCTGCTGCTGCTGCTGGCTGCTGCACTGGCACCTACTCAGACTCGG   1  M  V  P  C  T  L  L  L  L  L  A  A  A  L  A  P  T  Q  T  R  61 GCCTCAAAAAGTTACCCCTACGATGTCCCCGACTACGCCAGGACCGTGCCCCCTATGGTG  21  A  S  K  S  Y  P  Y  D  V  P  D  Y  A  R  T  V  P  P  M  V 121 CAGGTCACACGCTCAGAAGCTAGCGAGGGCCAAATCACCGTGACATGCCGAGCATCTGGG  41  Q  V  T  R  S  E  A  S  E  G  Q  I  T  V  T  C  R  A  S  G 181 TTCTATCCTTGGAACATTAACCTGAGTTGGAGGCAGGACGGGGTGTCCCTGTCTCACGAT  61  F  Y  P  W  N  I  N  L  S  W  R  Q  D  G  V  S  L  S  H  D 241 ACTCAGCAGTGGGGCGACGTGCTGCCAGATGGCAATGGGACCTACCAGACATGGGTGGCT  81  T  Q  Q  W  G  D  V  L  P  D  G  N  G  T  Y  Q  T  W  V  A 301 ACTCGGATCTCCCAGGGGGAGGAACAGAGATTCACCTGCTATATGGAGCATAGTGGACAG 101  T  R  I  S  Q  G  E  E  Q  R  F  T  C  Y  M  E  H  S  G  Q 361 CACTCAACACATCCTGTGCCATCTGGCAAGGTGCTGGTCCTGCAGAGTCACTGGCAGACA 121  H  S  T  H  P  V  P  S  G  K  V  L  V  L  Q  S  H  W  Q  T421 TTTCATTGA (SEQ ID NO: 19)  141  F  H  *  (SEQ ID NO: 20) Restriction analysis on DNAMAN1  Methylation: dam-No dcm-No Screened with 117 enzymes, 5 sites found  (SEQ ID NO: 21)BspMI  2 ACCTGCNNNN/       343   111  (SEQ ID NO: 22)Eam1105I 1 GACNNN/NNGTC       222  NheI  1 G/CTAGC       139 PstI  1 CTGCA/G       404  List by Site Order 111 BspMI 222 Eam1105I 343 BspMI 404 PstI  139 NheI  Non Cut Enzymes AatII  Acc65I   AccIII  AclI     AflII  AgeI AhaIII Alw441   AlwNI   ApaBI    ApaI   ApaLI AscI   Asp718I  AsuII   AvrII    BalI   BamHI BbeI   BbvII    BolI    BglI     BglII  Bpu1102I Bsc91I BsiI     BsmI    Bsp1407I BspHI  BspMII BssHII BstD102I BstEII  BstXI    Bsu36I ClaI Csp45I CspI     CvnI    DraI     DraIII DrdI EagI   Ecl136II Eco31I  Eco47III Eco52I Eco56I Eco57I Eco72I   EcoICRI EcoNI    EcoRI  EcoRV EheI   EspI     FseI    HindIII  HpaI   I-PpoI KpnI   MfeI     Mlu113I MluI     MscI   MstI MstII  NaeI     NanI    NcoI     NdeI   NotI NruI   NsiI     PacI    PflMI    PinAI  PmaCI PmeI   PvuI     PvuII   RleAI    SadI   SacII Sall   SapI     SauI    ScaI     SciI   SfiI SgrAI  SmaI     SnaBI   SpeI     SphI   SplI SpoI   SrfI     SspI    SstI     SstII  StuI SunI   SwaI     Tth111I VspI     XbaI   XcmI XhoI   XmaI     XmaIII  XmnI     XorII  4099: MICA002_baculo_BglIIfor (SEQ ID NO: 23) 5′ AAAAAAAGATCTATGGTCCCCTGTACCCTGCTGCTGCTGC 3′  4100: MICA002_baculo_EcoRIRev  (SEQ ID NO: 24)5′ AAAAAAGAATTCTCAATGAAATGTCTGCCAGTGACTCTGC 3′  

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A vaccine composition comprising, as animmunogenic component, an effective amount of a peptide comprising orconsisting of the MIC alpha 3-domain, the effective amount being anamount effective to elicit an immune response against the MIC alpha3-domain.
 2. The vaccine composition of claim 1, wherein the MIC alpha3-domain is a MICA or MICB alpha 3-domain.
 3. The vaccine composition ofclaim 1 or 2, wherein the MIC alpha 3-domain is non glycosylated.
 4. Thevaccine composition of any one of claims 1-3, wherein the peptidecomprises or consists of the amino acid sequence of SEQ ID NO: 3 or SEQID NO: 4, or an amino acid sequence that is at least 90% identicalthereto.
 5. The vaccine composition of any one of claims 1-4, whereinthe vaccine composition comprises a plurality of peptides.
 6. Thevaccine composition of any one of claims 1-5, wherein the peptide isconjugated to carrier protein.
 7. The vaccine composition of claim 1further comprising GM-CSF.
 8. A fusion protein comprising a monomericferritin subunit protein joined to a MIC alpha 3-domain protein, whereinthe monomeric ferritin subunit protein comprises a domain that allowsthe fusion protein to self-assemble into nanoparticles.
 9. The fusionprotein of claim 8, wherein the monomeric subunit is a monomeric subunitof a Helicobacter pylori ferritin protein.
 10. The fusion protein ofclaim 8 or 9, further comprising a Cytosine-Guanosine (CpG)oligonucleotide sequence.
 11. A nanoparticle comprising the fusionprotein of any one of claims 8-10.
 12. A nanoparticle comprising aplurality of MIC alpha 3-domain peptides.
 13. A vaccine compositioncomprising the nanoparticle of claim 11 or
 12. 14. The vaccinecomposition of claim 13, further comprising GM-CSF.
 15. A method oftreating cancer in a subject, the method comprising administering to asubject a composition of any one of claim 1-13.
 16. The method of claim15, further comprising administering GM-C S F.
 17. The method of claim15 or 16, wherein the composition is administered as part of atherapeutic regimen.
 18. The method of claim 17, wherein the therapeuticregimen is radiation therapy, targeted therapy, immunotherapy, orchemotherapy.
 19. The method of any one of claims 15-18, wherein saidsubject has tested positive for shed MIC in their serum.
 20. The methodof any one of claims 15-19, comprising administering to the subject oneor more vaccines specific for an antigen other than a MIC alpha 3-domainantigen.
 21. A method for treating cancer comprising administering tosaid subject a vaccine comprising cells that express MIC alpha-3 domain.22. A method for treating cancer where an immune response against MIC isinduced by use of a replicating or non-replicating virus.
 23. A methodof preventing the progression of cancer, the method comprisingadministering to a subject a composition of any one of claims 1-14. 24.The method of claim 23, wherein preventing the progression of cancercomprises preventing metastasis of the cancer or delaying tumor growth.25. The method of claim 23, wherein said subject has tested positive forshed MIC in their serum.
 26. The method of claim 23, wherein the canceris associated with overexpression of MICA.
 27. A method of causingclinical regression of cancer, the method comprising administering to asubject a composition of any one of claims 1-14.
 28. The method of claim27, wherein said subject has tested positive for shed MIC in theirserum.
 29. The method of claim 27, wherein the cancer is associated withoverexpression of MICA.
 30. A method of eliciting an immune responseagainst the MIC alpha 3-domain, the method comprising administering to asubject a composition of any one of claims 1-14.
 31. The method of claim30, wherein the composition elicits an immune response against the MICalpha 3-domain, but not against the MIC alpha 1-domain or MIC alpha2-domain.
 32. The method of claim 30, wherein said subject has testedpositive for shed MIC in their serum.
 33. The method of claim 30,wherein the cancer is associated with overexpression of MICA.